Document [original]

Application of Surface Enhanced Raman

Spectroscopy to Biological Systems

vorgelegt von

Diplom-Chemiker

Hoang Khoa Ly

aus Hanoi

von der Fakult¨

at II – Mathematik und Naturwissenschaften

der Technischen Universit¨

at Berlin

zur Erlangung des akademischen Grades

Doktor der Naturwissenschaften

– Dr. rer. nat. –

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Reinhard Schom¨

acker

Gutachter: Prof. Dr. Peter Hildebrandt

Gutachter: Prof. Dr. Ulla Wollenberger

Tag der wissenschaftlichen Aussprache: 05. Oktober 2012

Berlin 2013

D 83

Mama und Papa

...đời đời không tàn với khúc nhạc lòng tôi

Abstract

In this thesis resonance Raman (RR) and surface enhanced resonance Raman (SERR) spectroscopy

were applied to investigate biological systems, particularly heme containing proteins. The electronic

transition of the heme group can be exploited so that, with a properly tuned light excitation, highly

intense Raman signals solely of the cofactor were obtained. If the proteins are adsorbed on a rough

silver surface, then the resonance effect can be combined with the surface enhancement effect to

yield a highly sensitive technique, which allows probing of molecules even at sub-monolayer cover-

age. Additionally, if the surface is constituted by an electrode, surface redox reactions and kinetics of

adsorbed molecules can be investigated.

Such a setup was used to study the electron transfer (ET) properties of cytochrome c(cytc), a small

soluble redox protein which acts as an electron shuttle in the respiratory chain. Cytcwas immobilized

on a rough silver electrode coated with a biomimetic monolayer of ω-carboxylated alkanethiols and

investigated by stationary and time resolved SERR spectroscopy. Specifically, the ET kinetics of the

protein at short distances to the electrode was monitored. The results provide new insights into the

heterogeneous protein ET and indicate a central role of the interfacial electric field that has influence

on the protein orientational dynamics as well as on the electronic coupling and the rearrangement of

hydrogen bond networks at the protein-monolayer interface.

Beside its function in bioenergetics, cytcalso plays a key role as a signal transducer in the apoptosis,

the programmed cell death. To exert this function, it has to be detached from the inner mitochondrial

membrane, where it is bound to under normal conditions and released to the cytosol. The factors that

control the transfer of cytcare still under debate. In this respect a possible influence of posttransla-

tional Tyr nitration to switch cytc’s function from ET to peroxidation, which is considered to be the

first step in initiating apoptosis, is discussed. Therefore, mono-tyrosine and nitrated mono-tyrosine

mutants were investigated using RR spectroscopy in order to evaluate the impact of tyrosine nitration

on the integrity of the heme pocket. It could be shown that the destabilization caused by nitration is

not directly correlated with the respective peroxidase activity of the mutant. Moreover, experiments

in which the mutants were immobilized on a monolayer coated electrode showed that the destabiliza-

tion, promoted by the interfacial electric field, did not depend on the nitration.

Furthermore, the applicability of time resolved SERR spectroscopy for kinetic investigations of

biofilms of the bacterium Geobacter Sulfurreducens, grown on rough silver electrodes, was demon-

strated. The bacterium possesses a large number of multi-heme proteins, so-called outer membrane

cytochromes (Omcs), which facilitate the extracellular ET. These cytochromes are accumulated at the

biofilm-electrode interface and therefore can be probed using SERR spectroscopy. By applying sta-

tionary and time resolved SERR spectroscopy, it could be shown that these cytochromes function as

electron ’gates’ between the biofilm and the electrode, and an average heterogeneous ET rate constant

could be determined. Finally, results obtained from kinetic simulations indicate that the interfacial

ET is the bottleneck of the biofilm-electrode ET process.

The last part of the thesis is dedicated to methodological developments to expand the applicability of

surface enhanced Raman (SER) spectroscopy to non-coinage metals. Here, a Pt-Ag device was fab-

ricated, using a nanostructured Ag support which was coated by a dielectric layer and subsequently

covered by a Pt island film. The idea is that the isolated underlying Ag support should provide the

necessary surface enhancement, while the top metal layer promotes the interfacial reactions to be

studied. The fabricated hybrid device was tested in terms of stability and SER performance.

III

Zusammenfassung

In dieser Arbeit wurden die Resonanz-Raman-Spektroskopie (RR-Spektroskopie) sowie die

oberfl¨

achenverst¨

arkte Resonanz-Raman-Spektroskopie (SERR-Spektroskopie) verwendet, um

biologische Systeme, insbesondere H¨

amproteine, zu untersuchen. Der elektronische ¨

Ubergang

der H¨

amgruppe kann ausgenutzt werden, um ¨

uber gezielte Lichtanregung ausschließlich stark

verst¨

arkte Raman-Streusignale des Kofaktors zu erhalten. Adsorbiert an aufgerauten Silberelektro-

den kommt der Oberfl¨

achenverst¨

arkungseffekt zum Tragen, der es erm¨

oglicht, Proteine selbst bei

Submonolagen-Beschichtung zu untersuchen. Dar¨

uber hinaus erm¨

oglicht die Elektrode die Ans-

teuerung von Oberfl¨

achenredoxreaktionen, was ausgenutzt werden kann, um Informationen ¨

uber die

Redox-Eigenschaften und Dynamik von H¨

amproteinen zu erhalten.

Dieser experimentelle Ansatz wurde verwendet, um die Elektronentransferkinetik des Proteins

Cytochrom c- ein kleines, redox-aktives H¨

amprotein in der mitochondrialen Atmungskette - zu

untersuchen. Cytochrom cwurde daf¨

ur auf einer rauen Silberelektrode, beschichtet mit einer

biomimetischen Monoschicht aus carboxyl-terminierten Alkanthiolen, immobilisiert und mit sta-

tion¨

arer und zeitaufgel¨

oster SERR-Spektroskopie untersucht. Besonderes Augenmerk wurde auf

die Elektronentransfereigenschaften des Proteins bei kleinen Abst¨

anden zur Elektrode gelegt. Die

Ergebnisse der Untersuchungen geben neue Aufschl¨

usse ¨

uber den heterogenen Elektronentransfer

und deuten dabei auf eine besondere Rolle des elektrischen Feldes an Grenzfl¨

achen hin. Dieses

beeinflusst die Oberfl¨

achenorientierungsdynamik, die Elektronentunnel-Wahrscheinlichkeit und die

Umstrukturierung der Wasserstoffbr¨

uckennetzwerke des Protein und an der Grenzfl¨

ache.

Neben der Rolle als Elektronen¨

ubertr¨

ager in der Atmungskette fungiert Cytochrom cebenso als Sig-

nalprotein in der Apoptose, dem programmierten Zelltod. Daf¨

ur muss es die mitochondriale Mem-

bran, an die es normalerweise gebunden ist, verlassen und ins Zellplasma ¨

ubergehen. Die Ursache f¨

diese Migration ist noch weitestgehend ungekl¨

art. Als ein Faktor, der den damit verbundenen Verlust

an Redox- und die Zunahme an Peroxidase-Aktivit¨

at erkl¨

art, wird die post-translationale Nitrierung

von Tyrosinresten des Proteins diskutiert. Folglich wurde mittels RR-Spektroskopie der Einfluss

von post-translationaler Tyrosin-Nitrierung auf die Integrit¨

at der H¨

amtasche untersucht. Es zeigte

sich, dass die Destabilisierungen, hervorgerufen durch die Nitrierung verschiedener Tyrosinreste,

nicht mit der jeweiligen gesteigerten Peroxidase-Aktivit¨

at korrelieren. Experimente, bei denen die

verschiedenen Tyrosin-Mutanten auf eine Elektrode adsorbiert wurden, zeigten außerdem, dass die

Grenzfl¨

achen bedingte Destabilisierung der H¨

amtasche unabh¨

angig von der Nitrierung wirkt.

Des Weiteren wurde die Anwendbarkeit der zeitaufgel¨

osten SERR-Spektroskopie zur kinetischen

Untersuchung von Biofilmen des Bakteriums Geobacter Sulfurreducens demonstriert. Dies ist

m¨

oglich, da das Bakterium eine Reihe von Multih¨

amproteinen, sogenannten "outer membrane

cytochromes" (Omcs), besitzt, die f¨

ur den extrazellul¨

aren Elektronentransfer verantwortlich sind.

Besonders stark sind diese Cytochrome an der Grenzfl¨

ache zwischen Biofilm und Elektrode akku-

muliert. Durch Anwendung von station¨

arer und zeitaufgel¨

oster SERR-Spektroskopie konnte die

besondere Rolle der Omcs als "Elektronenschleuse" zwischen Biofilm und Elektrode nachgewiesen

und eine gemittelte heterogene Elektronentransferratenkonstante bestimmt werden. Weiter deuten

die Experimente zusammen mit kinetischen Simulationen darauf hin, dass der heterogene Elektro-

nentransfer der geschwindigkeitsbestimmende Schritt beim Elektronenaustausch Biofilm-Elektrode

ist.

Der letzte Teil der Arbeit ist der methodischen Entwicklung der oberfl¨

achenverst¨

arkten Raman-

Spektroskopie zur Ausdehnung auf Nicht-M¨

unzmetalle gewidmet. Dazu wurde eine Pt-Ag-Hybrid-

Zusammenfassung

Elektrode konstruiert, die aus einem nanoskopisch rauen Silberuntergrund, welcher zuerst mit

einem dielektrischen Material beschichtet und anschließend mit einem Platinfilm ¨

uberzogen wurde.

W¨

ahrend der abgeschottete Silberuntergrund f¨

ur die notwendige Oberfl¨

achenverst¨

arkung sorgt,

laufen an der Pt-Oberfl¨

ache die zu untersuchenden Grenzfl¨

achenreaktionen ab. Die Hybrid-

Elektrode wurde in Bezug auf ihre Stabilit¨

at und ihre Signalverst¨

arkungseigenschaften bei ver-

schiedenen Wellenl¨

angen untersucht. Die Resultate zeigen, dass starke Raman-Signale von Proben-

molek¨

ulen, adsorbiert auf der ¨

außeren Pt-Schicht, erhalten werden k¨

onnen. Die Signale sind nur

geringf¨

ugig schw¨

acher im Vergleich zu einer direkten Adsorption auf Ag.

Publications

Parts of this work are published in the following articles:

1. Ly, H. K.; Marti, M. A.; Martin, D. F.; Alvarez-Paggi, D.; Meister, W.; Kranich, A.; Weidinger,

I. M.; Hildebrandt, P.; Murgida, D. M. Thermal Fluctuations Determine the Electron-Transfer

Rates of Cytochrome c in Electrostatic and Covalent Complexes. ChemPhysChem 2010, 11(6),

1225–1235.

2. Ly, H. K.; Wisitruangsakul, N.; Sezer, M.; Feng, J. J.; Kranich, A.; Weidinger, I. M.; Zebger,

I.; Murgida, D. M.; Hildebrandt, P. Electric Field Effects on the Interfacial Electron Transfer

and Protein Dynamics of Cytochrome c. J. Electroanal. Chem. 2011, 660, 367–376.

3. Ly, H. K.; Sezer, M.; Wisitruangsakul, N.; Feng, J. J.; Kranich, A.; Millo, D.; Weidinger, I. M.;

Zebger, I.; Murgida, D. M.; Hildebrandt, P. Surface Enhanced Vibrational Spectroscopy for

Probing Transient Interactions of Proteins with Biomimetic Interfaces: Electric Field Effects

on Structure, Dynamics and Function of Cytochrome c. FEBS Review 2011, 278, 1382–1390.

4. Ly, H. K.; K¨

ohler, C.; Fischer, A.; Kabuß, J.; Schlosser, F.; Schoth, M.; Knorr, A.; Weidinger,

I. M. Induced Surface Enhancement in Coral Pt Island Films Attached to Nanostructured Ag

Electrodes. Langmuir 2012, 28, 5819–5825.

5. Ly, H. K.; Utesch, T.; Díaz-Moreno, I.; Garía-Heredia, J. M.; Rósa, M. A. d. l.; Hildebrandt,

P. Pertubation of the Redox Site Structure of Cytochrome c upon Tyrosine Nitration. J. Phys.

Chem. C 2012, 116, 5694–5702.

6. Ly, H. K.; Harnisch, F.; Hong, S.-F.; Schr¨

oder, U.; Hildebrandt, P; Millo, D. Unraveling the

Interfacial Electron Transfer Dynamics of Electroactive Microbial Biofilms using Surface-

Enhanced Raman Spectroscopy. ChemSusChem 2012; DOI: 10.1002/cssc.201200626.

VII

Publications

Other publications in peer-reviewed journals:

1. Ledesma, G. A.; Murgida D. H.; Ly, H. K.; Wackerbarth, H.; Ulstrup, J.; Costa-Filho, A. J.;

Vila, A. J. The Met Axial Ligand Determines the Redox Potential in CuA Sites. J. Amer. Chem.

Soc. Comm. 2007, 129(39), 11884–11885.

2. Wisitruangsakul, N.; Zebger, I; Ly, H. K.; Murgida, D. H.; Ekgasit, S.; Hildebrandt, P.

Redox-linked Protein Dynamics of Cytochrome c Probed by Time-Resolved Surface Enhanced

Infrared Absorption Spectroscopy. Phys. Chem. Chem. Phys 2008, 10, 5267–5268.

3. Kranich, A.; Ly, H. K.; Hildebrandt, P.; Murgida, D. M. Direct Observation of the Gating Step

in Protein Electron Transfer: Electric-Field-Controlled Protein Dynamics. J. Am. Chem. Soc.

2008, 130, 9844–9848.

4. Millo, D.; Ranieri, A.; Gross, P.; Ly, H. K.; Borsari, M.; Hildebrandt, P.; Wuite, G. J. L.;

Gooijer, C.; Zwan, G. v. d. Electrochemical Response of Cytochrome c Immobilized on Smooth

and Roughened Silver and Gold Surfaces Chemically Modified with 11-Mercaptounodecanoic

Acid. J. Phys. Chem. C 2009, 113(7), 2861-2866.

5. Sezer, M.; Feng, J. J.; Ly, H. K.; Shen, Y; Nakanishi, T.; Kuhlmann, U.; Hildebrandt, P.;

M¨

ohwald, H.; Weidinger, I. M. Multi-layer Electron Transfer Across Nanostructured Ag-SAM-

Au-SAM Junctions Probed by Surface Enhanced Raman Spectroscopy. Phys. Chem. Chem.

Phys 2010, 12, 9822–9829.

6. Hildebrandt, P.; Feng, J. J.; Kranich, A.; Ly, H. K.; Martin, D. F.; Martin, M.; Murgida, D. H.;

Alvarez-Paggi, D.; Wisitruangsakul, N.; Sezer, M.; Weidinger, I. M.; Zebger, I. Electron Trans-

fer of Proteins at Membrane Models. In Surface Enhanced Raman Spectroscopy: Analytical,

Biophysical and Life Science Applications; Schl¨

ucker, S., Ed.; Wiley-VCH, 2010; 219–241.

7. Millo, D.; Harnisch, F.; Patil, S. A.; Ly, H. K.; Schr¨

oder, U.; Hildebrandt, P. In situ Spec-

troelectrochemcial Investigation of Electrocatalytic Microbial Biofilms by Surface-Enhanced

Resonance Raman Spectroscopy. Angew. Chem. Int. Ed. 2011, 123(11), 2673-2675.

8. Sivanesan, A.; Ly, H. K.; Kozuch, J.; Sezer, M.; Kuhlmann, U.; Fischer, A.; Weidinger, I. M.

Functionalized Ag Nanoparticles with Tunable Optical Properties for Selective Protein Analy-

sis. Chem. Commun. 2011, 47, 3553–3555.

9. Sivanesan, A.; Kozuch, J.; Ly, H. K.; Kalaivani, G.; Fischer, A.; Weidinger, I. M. Tailored

Silica Coated Ag Nanoparticles for Non-Invasive Surface Enhanced Raman Spectroscopy of

Biomolecular Targets. RSC Adv 2012, 2, 805–808.

VIII

Talks:

1. Electron transfer kinetics of electrostatically and covalently immobilized horse heart

Cytochrome c on OH/COOH terminated SAMs, Invited Lecture at: Workshop on Structure,

dynamics, and function of proteins. 2008 Sept. 23 - 25; ITQB Oeiras, Portugal - Technical

University of Berlin/ UNICAT - Unifying Concepts in Catalysis..

Poster contributions:

1. Sezer, M.; Ly, H. K.; Feng, J.-J.; Kranich, A.; Utesch, T.; Mroginski, M. A.; Kuhlmann, U.;

Murgida, D. H.; Hildebrandt, P.; Weidinger. I. M. Electron Transfer Properties of Cytochrome

c on Ag Electrodes Measured with Surface Enhanced Resonance Raman Spectroscopy. Poster

at: Tagung der Deutschen Gesellschaft f¨

ur Biophysik; 2008 Sept. 28 - Oct 01.; Freie Universit¨

Berlin, Germany.

2. Ly, H. K.; Fischer, A.; Hildebrandt, P.; Weidinger, I. M.; Induced SER Activity in Ag-Pt

Hybrid Devices. Poster at: International Conference on Raman Spectroscopy; 2010 Oct. 08

- 15, Boston, MA, USA.

3. Ly, H. K.; Harnisch, F.; Hong, S-F; Schr¨

oder, U.; Hildebrandt, P.; Millo, D. Kinetics of the Het-

erogeneous Electron Transfer of Outer Membrane Cytochromes in Electrocatalytic Biofilms

Studied by Time Resolved Surface Enhanced Resonance Raman Spectroscopy. Poster at: Inter-

national Microbial Fuelcells Conference, 2011 June 06 - 08; Leeuwarden, The Netherlands.

4. Ly, H. K.; Fischer, A.; K¨

ohler, C.; Kabuß, J.; Schlosser, F.; Knorr, A.; Hildebrandt, P.; Wei-

dinger, I. M. SER active Pt-Ag Hybrid Device for Probing Pt-Catalyzed Reactions. Poster at:

International Symposium on Relations between Homogeneous and Heterogeneous Catalysis.

2011 Sept 11 - 16; Freie Universit¨

at Berlin, Germany.

Contents

Abstract III

Zusammenfassung V

Publications VII

Contents XI

1 Introduction and Motivation 1

2 Materials and Methods 5

2.0.1 RamanEffect.................................. 5

2.0.2 Quantum Mechanical Treatment of the Raman Effect . . . . . . . . . . . . . 6

2.0.3 Resonance Raman Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.0.4 Surface Enhanced Raman . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.0.5 Surface Enhanced Resonance Raman . . . . . . . . . . . . . . . . . . . . . 12

2.1 Properties of Electrochemical Interfaces . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1.1 Self Assembled Monolayer - SAM . . . . . . . . . . . . . . . . . . . . . . . 14

2.2 Hemeproteins...................................... 15

2.2.1 Cytochromes .................................. 16

2.2.2 Resonance Raman Spectroscopy of Cytochromes . . . . . . . . . . . . . . . 18

2.3 ElectronTransferTheory................................ 20

2.3.1 Homogeneous ET and Classical Marcus Theory . . . . . . . . . . . . . . . . 20

2.3.2 HeterogeneousET ............................... 23

3 Experimental Details and Instrumentation 25

3.1 Confocal Raman Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2 Electrochemical Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.3 Resonance Raman and Surface Enhanced Resonance Raman Measurements . . . . . 26

4 Results 29

4.1 Electron Transfer Properties of Cytochrome cat Electrochemical Interfaces . . . . . 29

4.2 Tyrosine Nitrated Cytochrome c- Role in Apoptosis . . . . . . . . . . . . . . . . . . 33

4.3 Interfacial ET of Outer Membrane Cytochromes Embedded in Biofilms of Geobacter 35

4.4 Surface Enhanced Raman Active materials - Pt Coated Ag Electrodes . . . . . . . . 41

5 Conclusions and Outlook 43

Bibliography 47

Contents

6 Thermal Fluctuations Determine the Electron-Transfer Rates of Cytochrome cin Elec-

trostatic and Covalent Complexes 63

7 Electric Field Effects on the Interfacial Electron Transfer and Protein Dynamics of

Cytochrome c77

8 Perturbation of the Redox Site Structure of Cytochrome cupon Tyrosine Nitration 91

9 Unraveling the Interfacial Electron Transfer Dynamics of Electroactive Microbial

Biofilms Using Surface-Enhanced Raman Spectroscopy 103

10 Induced Surface Enhancement in Coral Pt Island Films Attached to Nanostructured Ag

Electrodes 113

XII

Abbreviations

5c five coordinated

5cHS five coordinated high spin

6c six coordinated

6cHS six coordinated high spin

6cLS six coordinated low spin

BES bioelectrochemical systems

CCD charged coupled device

CE couter electrode

CV cyclic voltammetry

Cytc Cytochrom c

DOS density of states

EC electrochemistry

ET electron transfer

FC Franck-Condon

FCWD Franck-Condon weighted density of states

HS high spin

LS low spin

MFC microbial fuel cells

MO molecular orbital

MUA 11-mercaptoundecanoic acid

NHE normal hydrogen electrode

Omc outer membrane cytochrome

RE reference electrode

RR resonance Raman

RT room temperature

SAM self assembled monolayer

SEIRA surface enhanced infrared absorption

SER surface enhanced Raman

SERR surface enhanced resonance Raman

UV/Vis ultraviolet / visible

WE working electrode

XIII

1 Introduction and Motivation

Resonance Raman (RR) and surface enhanced Raman (SER) are vibrational spectroscopic methods

of high sensitivity. While resonance Raman spectroscopy is suitable to probe molecules that exhibit

an electronic transition matching the excitation laser line, surface enhanced Raman spectroscopy

focusses on molecules in vicinity of metal surfaces that allow the optical excitations of surface plas-

mons. In both cases, the obtained signal intensities are very high and can exceed those of ordinary

Raman spectroscopy by several orders of magnitude.

In the 1970th, Spiro et al successfully employed resonance Raman spectroscopy for the investigation

of chromophore containing proteins. These were specifically heme containing proteins such as the

cytochromes, the cytochrome c oxidase and the cytochrome P450 but also the oxygen transporting

proteins myo- and hemoglobin. The advantage of resonance Raman spectroscopy is that, if the exci-

tation laser line is tuned to match an electronic transition of the chromophore, then only vibrational

bands originating from the cofactor will be obtained. Hence, signal interferences from the protein

backbone as in IR absorption experiments are negligible. Thus, this method represents a versatile

technique for detailed investigation of constitution and functioning of a cofactor.

The best investigated protein in this context so far is the small soluble heme protein cytochrome c.

This protein is found in aerobic organisms where it is located at the inner mitochondrial membrane

and plays a central role in the respiratory chain. It acts as an electron carrier protein and shuttles

electrons between the integral membrane enzymes cytochrome creductase and cytochrome coxdiase

(complex III and complex IV, respectively). Moreover, recent studies also point to a crucial role of

cytochrome cin the mitochondrial mediated apoptosis, revitalizing the discussion about this protein.

In many studies using also various other spectroscopic and electrochemical methods, cytochrome

cwas used as a model compound to gain general insights into biological electron transfer (ET)

processes. A common experimental setup to probe electron transfer kinetics of cytochrome ccon-

sists of a solid metal electrode coated with self-assembled monolayers to promote an adsorption

of the protein in its native state. Redox reactions are triggered by changing the applied potential

either stepwise or in a potential-jump mode. The employment of solid electrodes inheres numerous

advantages compared to studies in solution. The electrode-SAM system can be adapted to mimic

biological membranes where most electron transfer reactions occur. For cytochrome c, a monolayer

consisting of negatively charged ω-carboxylated alkanethiols has turned out to be most suitable.

However, the adsorption of cytochrome con such monolayers yields coverages in the order of

picomol per cm2, which is even below a monolayer coverage. Under these conditions even resonance

Raman spectroscopy reaches its limits. Therefore, to boost the signal intensity dramatically, a combi-

nation of resonance Raman and surface enhanced Raman is required, i.e. surface enhanced resonance

Raman (SERR) spectroscopy. This technique utilizes a rough metal support that can produce surface

plasmon resonances upon light excitation. These plasmons are quantized vibrations of conducting

electrons within the metal, which, in turn, generate strongly oscillating electric fields in the near-

field of the surface. These enhanced electric fields are commonly believed to be responsible for a

drastically increase in Raman scattering cross section of molecules in close vicinity to such a metal

surface. To combine resonance Raman and surface enhanced Raman spectroscopy, an excitation line

has to be selected that is in resonance with both the electronic transition of the chromophore of the

adsorbed molecule and the plasmons of the underlying metal support. The required wavelength in

the latter case varies strongly with the kind of metal due to the different electronic properties. For

cytochromes exhibiting a strong electronic absorption in the near UV region at ca. 400 nm (Soret

1. Introduction and Motivation

band), nanoscopically rough Ag surfaces match both conditions. Upon using such nanostructured

Ag electrodes coated with an appropriate SAM, high intensity signals of cytochrome care obtained.

The vibrational band pattern of heme proteins is well known and undergoes only slight changes

upon adsorption. Therefore, it is possible to draw conclusion about the spin, ligation and oxidation

state of the adsorbed proteins by analyzing the spectra. Such an approach was successfully applied

by Hildebrandt et al to study the redox and conformational kinetics and equilibria of adsorbed

cytochrome cand other redox enzymes.

In addition to biological applications of SE(R)R spectroscopy, there is an enormous potential of

non-resonant surface enhanced Raman (SER) and SERR spectroscopy in surface an interfacial

science in general, such as in heterogeneous catalysis or photovoltaic. To optimize such processes, a

profound knowledge about the underlying molecular mechanisms is required. In principle, this can

be provided by SER spectroscopy. The only drawback is related to the metal support. Unfortunately,

most technically relevant reactions require metals or materials (e.g. Pt, Rh or MgO and TiO2) that

provide only low or no intrinsic surface enhancements. Consequently, those metals that exhibit high

surface enhancements (Ag and Au) are of lower technological interests. A possible solution for this

problem are hybrid metal composites, consisting of a supporting metal with high field enhancements

covered by a layer of metal of choice for a certain technical application. Although this principle is

known for a long time, the exploitation of this synergetic effect, provided by metals with different

electronic properties, is about to be investigated more systematically. Real applications to relevant

technical processes are yet still missing.

In the first part of this thesis, surface enhanced resonance Raman spectroscopy with Soret band

excitation was applied to investigate the electron transfer kinetics of horse heart cytochrome c

adsorbed on an Ag electrode coated with a carboxyl-terminated alkanethiol SAM. Additionally, Q

band excitation methods, which allow probing of the orientational dynamics and kinetics of the pro-

tein, were employed. In combination with findings obtained by other methods such as theoretical

calculations, cyclic voltammetry and surface enhanced infrared absorption spectroscopy, the results

provide novel insights into the electric field dependence of the various steps of the interfacial redox

process.

In the second part, RR and SERR spectroscopy was used to study the effects of tyrosine nitration on

the heme site structure of human heart cytochrome c. Tyrosine nitration and other posttranslational

protein modifications are believed to play a key role in altering the function of cytochrome cfrom an

electron shuttle protein to a crucial signal transducer in the apoptotic reaction cascades. Information

on the effects of different tyrosine nitrations was obtained by using mono-tyrosine and nitrated mono-

tyrosine mutants. Immobilizing the protein variants on electrodes coated with biomimetic membrane

models allows studying the combined effects of Tyr nitration and local electric fields on the structural

and functional properties of the protein.

In the third part of this work, the application of time resolved SERR spectroscopy for determining ET

kinetics of outer membrane cytochromes (Omcs) embedded in electro-catalytically active biofilms of

Geobacter sulfurreducens grown on rough Ag electrodes is demonstrated. These microbes are metal-

reducing bacteria that can consume small organic compounds such as lactate or acetate to generate

electrons, which - if present - can be delivered to a solid terminal electron acceptor. Omcs are either

integral membrane or peripherally bound proteins including several heme cofactors to provide ET

routes from the cell to an external acceptor, in this case the electrode. After demonstrating the appli-

cability of SERR spectroscopy to probe redox processes of the Omcs, time resolved SERR studies

were carried out to monitor the ET dynamics of the Omcs directly. The results were discussed in

terms of an overall ET mechanism.

In the last part of this thesis, a Pt-Ag hybrid electrode was manufactured and systematically investi-

gated in terms of SER signal enhancements. Following a procedure proposed by Feng et al, a rough

silver electrode was covered with different dielectric spacers, and subsequently a platinum island film

1. Introduction and Motivation

was placed on top via reductive metallation. The signal performance was tested using cytochrome c

and 2-mercaptopyridine as probe molecules for 413 nm and 514 nm excitation, respectively.

2 Materials and Methods

In this chapter, the materials and the fundamentals of the applied methods are briefly introduced. This

includes a short description of Raman, resonance Raman and surface enhanced Raman spectroscopy.

Furthermore, an overview of the properties of electrochemical interfaces and a short introduction to

heme proteins, particularly cytochromes, are provided. Finally, a brief summary of electron transfer

theory is presented.

2.0.1 Raman Effect

The discussion on light scattering effects can be traced back to the times of Leonardo Da Vinci who

ascribed the blue color of the sky to light scattering on suspended macroparticles in the atmosphere

such as dusk, water droplets and ice crystals [1]. It took a long time until in 1873 Maxwell came to

the conclusion that it might be the molecule itself who acts as scattering center and thus, information

on the molecule’s properties are inherent in the scattered radiation. Although the first experimental

evidences for that were already stated by Lallemand in 1869, it was Lord Rayleigh who provided

a profound survey on that issue in 1899 [2]. The first indication for the existence of frequency

modified scattered radiation was stated by Smekal in 1923 [3]. In his theoretical work, he found that

upon photon induced transition of a molecule from one quantum state to another, the scattered light

contains frequencies that are smaller and larger than the frequency of the incident light by the energy

difference between the states. This observation could be experimentally proven by C. V. Raman in

1928 and independently by Landsberg and Mandelstam [4, 6]. Raman was awarded the Nobel prize

for his discovery. The frequency shift of a scattered radiation with respect to the incident radiation

due to energy transfer with molecules is referred to as Raman effect [5].

A classical approach to the theoretical fundamentals of Raman scattering is based on first order

induced electric dipoles. These are generated upon interaction between the oscillating electric field

vector Eof the incoming light and the electron cloud of the scattering center. The magnitude and

direction of the induced dipole moment vectors pdepend on the polarizability of the molecule. This

correlation is expressed as

p=α·E.(2.1)

αdenotes the 3x3 polarizability tensor and depends on the positions of the nuclei with respect to

the electron cloud and thus, is also sensitive to changes of the molecule’s internal coordinates. Each

non-linear molecule with Natoms possesses 3N-6 normal modes Qkwhich describes the oscillations

of each atom with the frequency ωk. The time dependence of Qkis given by

Qk(t) = Qk0cos(ωkt+δk).(2.2)

Qk0and δkdenote the amplitude and the phase factor of the vibration, respectively. The change of

the tensor components of αwith the molecular vibration along Qkcan be estimated using a Taylor

series

αρσ =αρσ 0+∑

k∂αρσ

∂Qk0

Qk+.... (2.3)

2. Materials and Methods

Figure 2.1: Jablonski diagram of different vibrational transitions induced by photon scattering. S0(Qk)

and S1(Qk) denote the electronic ground and the first excited singlet state, respectively. v

= 0 indicates the vibrational ground state. |riis the virtual state. ARayleigh scattering. B

Stokes Raman scattering. CAnti-Stokes Raman scattering. DResonance Stokes Raman

scattering.

ρand σdenote the molecules fixed internal coordinates and replace the Cartesian variables x,y

and z. The time dependence of αis then given by the combination of 2.2 and 2.3,

αk=α0+∂αρσ

∂Qk0

Qk0cos(ωkt+δk).(2.4)

For a plane electromagnetic wave represented by E=E0cos(ω1t), the induced dipole moment

vector is given by

p=α0E0cos(ω1t)+ 1

2∂αρσ

∂Qk0

E0Qk0cos(ω1±ωk±δk).(2.5)

The first term in equation 2.5 represents the Rayleigh scattering, which depends on the polarizabil-

ity of the molecule at nuclei equilibrium positions. The two last terms refer to the Raman scattering.

Accordingly, the scattered radiation is frequency shifted by ωk- the frequency of the kth normal

mode. Furthermore, the equation states that to obtain Raman scattered radiation the polarizability

tensor αhas to change with Qk,i.e. at least one component of the tensor ∂αρσ

∂Qk0has to be non-zero.

In general, the classical theory is able to describe the frequencies of scattered radiation but fails with

respect to the intensity of the scattered light. Moreover, it allows no conclusion on the molecule’s

properties. This information is only accessible by means of quantum theory.

2.0.2 Quantum Mechanical Treatment of the Raman Effect

The general quantum mechanical treatment of the two photon involving Raman scattering process

is based on second order perturbation theory and was first derived by Placzek on the basis of

Kramers and Heisenberg’s dispersion theory [9–11]. In this way, the molecule is described by quan-

tum mechanics while the radiation is treated classically. As a result, the Raman scattering originates

2. Materials and Methods

from a transition of the molecule from a vibrational quantum state |iito another vibrational quantum

state |fiwith the energies Eiand Ef, respectively. The transition is not direct but involves a virtual

state |riwith the energy Er. Figure 2.1 depics different possible transitions with different positions

of Ei,Efand Errelative to each other. The intensity of Raman bands which originate from such a

transition is proportional to following quantities

Ifi ≈I1exp−Ei

kbT(ω1±ωfi)4∑

ρσ αρσ 

fi.(2.6)

I1and ω1are the intensity and the frequency of the incident radiation, respectively. ωfi denotes

the frequency of the energy difference of |fiand |ii. The exponential term represents the Boltzmann

distribution and determines the number of possible scattering centers in state |iias a function of

temperature. αρσ f i denotes the respective ρσ element of the transition polarizability tensor. This

tensor is obtained from the solution of pfi =Dψ1

fˆpψ0

i+Dψ0

fˆpψ1

iby comparison with the

classical case described in equation 2.1 and together with the transition dipole moment replace the

oscillating electric dipole and the polarizability from the classical treatment. In general, the elements

of the transition polarizability tensor after Placzek can be calculated after

αρσ i f =1

h∑

r6=i,fhf|ˆpρ|rihr|ˆpσ|ii

ωri −ω1−iΓr

+hf|ˆpσ|rihr|ˆpρ|ii

ωr f +ω1+iΓr.(2.7)

ˆpρ,σdenote the dipole operator in σand ρdirection, respectively. ¯

hωri,¯

hωr f and ¯

hω1represent the

energy necessary for the transition from |rito |ii, from |rito |fiand the energy of the incident light,

respectively. The energy of the scattered radiation is ¯

h(ω1−ωfi) = ¯

hωs.iΓraccounts for the life time

of the state |ri. The sum is made over all possible intermediate states |ri. In principle, no restrictions

on the energy Erof these states are made and they can lay either below Eior above Ef. For normal

Raman scattering where ω1<< ωri, the value of αρσ f i is therefore determined by the weighted

normal Raman scattering can be regarded as a transition from |iito |riand |rito |fi, involving all

possible states |ri, where the transition dipole moment between the states |rito both |iias well as |fi

is non-zero.

2.0.3 Resonance Raman Effect

In case of resonant Raman scattering, the frequency of the exciting light ω1approaches an electronic

transition with the frequency ωri (i.e. |ri=|eiwhere |eidenotes an excited electronic state), see

figure 2.1 D. Then, the transition polarizability tensor αρσ f i is mainly dominated by one term of

the sum where ωri −ω1−iΓr≈−iΓris very small and additionally, hf|ˆpρ|rihr|ˆpσ|iibecomes very

large. This strongly increasing term is the resonant term. Accordingly, αρσ fi is reduced to

αρσ f i =1

h∑

r=fhf|ˆpρ|rihr|ˆpσ|ii

ωri −ω1−iΓr.(2.8)

Since an electronic transition is involved, it is convenient to employ the Born-Oppenheimer

approximation, which allows to separate the nuclear wave functions from those of the electrons,

yielding |ii=|vii|gi,|fi=vf|giand |ri=|ni|ei.|giand |eidenote the electronic ground and

the first excited state while |vii,vfand |nirepresent the initial, final and virtual vibrational states,

respectively. Under this simplification, each integral can be separated into two parts according to

hvinipge,ρand nvfpge,σ, and the transition polarizability is further simplified to

2. Materials and Methods

αρσ f i =1

h∑

r(hvinipge,ρnvfpge,σ

ωri −ω1−iΓe),(2.9)

where pge,ρand pge,σdenote the electronic transition dipole moments in ρand σdirection, respec-

tively. To account for the dependence of pρ,σon the nuclear vibrations Qk, the transition dipole

moments are expanded in a Taylor series around the displacement from the equilibrium position Q0,

yielding

pge,ρ(Qk) = p0

ge,ρQ0

k+∑∂pge,ρ

∂Qkk

Qk+... (2.10)

pge,σ(Qk) = p0

ge,σQ0

k+∑∂pge,σ

∂Qkk

Qk+.... (2.11)

ge,ρQ0

kdenotes the transition dipole moment at nuclei equilibrium positions. Upon combination

of equation 2.9 and 2.11, the transition polarizability can be divided in different terms that represent

different contribution mechanisms to resonance Raman scattering.

αρσ f i =Aρσ +Bρσ +Cρσ (2.12)

with

Aρσ =1

hp0

ge,ρp0

ge,σ∑hvininvf

ωri −ω1−iΓer

(2.13)

Bρσ =1

h p0

ge,ρpge,σ∑hvi|Qk|ninvf

ωri −ω1−iΓer!(2.14)

... +p0

ge,σpge,ρ∑hvinihn|Qkvf

ωri −ω1−iΓer

(2.15)

Cρσ =.... (2.16)

The Aρσ term contains two Franck-Condon (FC) integrals. Accordingly, the contribution of this

term to resonance enhancement is stronger with decreasing orthogonality character of the wave func-

tions in the ground and the exited state. This is particular the case for total symmetric vibrational

modes that are coupled to strong electronic transitions such as σ−σ∗or π−π∗. Those modes gain

most of their intensity via the Aρσ term.

The Bρσ term arises from the Herzberg-Teller vibronic coupling, which is expressed by p0

ρ,σfor small

displacements of Qk. There, two or more electronic states are coupled as a consequence of mixing of

the respective Hamiltonians (i.e. perturbation of the respective Hamiltonian due to vibration). It can

be shown that the resulting transition dipole moment of a transition of from |gito |eimay contain

(borrow) also contributions from transitions originating from |gito another nearby excited electronic

state |hi. For the kth vibration, the amount of the ’borrowed’ transition dipole moment is proportional

to the appropriate coupling integral and the displacement Qk. Furthermore, the coupling is higher,

the smaller the energy difference of the coupled exited states is. The Bρσ term scattering can account

for the RR activity of total symmetric and non-total symmetric modes. However, due to parity selec-

tion rules, the Bρσ term enhancement of total symmetric modes require the coupling of electronic

excited states with same symmetry. This condition is usually not matched. Furthermore, Bρσ term

scattering becomes most pronounced when the ω1matches a weak electronic transition that is in turn

2. Materials and Methods

vibronically coupled with a nearby strong one. The Cρσ terms considers overtones, which are usually

not well pronounced.

2.0.4 Surface Enhanced Raman

Surface enhanced Raman (SER) scattering denotes the significant Raman signal enhancement of

probe molecules close to rough metal surfaces. Depending on the setup of the experiment, the

enhancement can exceed ordinary Raman signal intensities by a factor of 106and more [17–19].

Thus, after the first observation by Fleischmann et al, in 1974, the potential of this effect was soon

recognized [15, 16]. Since then, SER spectroscopy has been continuously refined and has become a

powerful tool to probe structures and dynamics even at a nanomolar concentration level. Inspired by

the work of Nie et al as well as Kneipp et al who showed that under proper chosen conditions, surface

enhancement can be strong enough to detect even Raman signals of single molecules, the field of

SER spectroscopy has gained a lot of scientific ascent lately [20, 21]. Finally, SER spectroscopy has

also put a lot of momentum to other fields of science such as nanotechnology, nanodesign, near-field

optics and last but not least gave birth to the field of plasmonics [22, 23].

The surface enhancement (SE) effect can be largely understood on the basis of classical elec-

tromagnetic theory, as proposed by Moskovitz and Kerker et al [24–26]. The general idea is the

generation of dipolar surface plasmon resonances upon light irradiation on a metal (particle) surface.

Surface plasmons are collective plasmonic vibrations of free electrons with respect to the nuclei,

parallel to the metal surface. For a particle, which size is small with respect to the wavelength of the

incident light with a frequency ω1, the local (or near-) field is given by

Eloc (ω1) = Einc (ω1)+Edipole (ωp).(2.17)

Einc (ω1)and Edipole (ω1)denote the electric field vector of the incident light and the field vector

due to induced dipole moments, respectively. The total induced dipole moment ppin a particle is

according to classical theory pp=αpEloc. The polarizability αpdepends mainly on the kind of

metal and is connected to its characteristic dielectric function ε(ω)by

αp=R3ε−1

ε+2.(2.18)

Rdenotes the radius of the metal particle. The dielectric function is frequency dependent and is,

considering interband transitions, given by

ε=εb+1−ω2

ω2+iωγ ,(2.19)

where εbdenotes the frequency dependent contribution of interband transitions to the dielectric

function and ωpand γare the metal’s plasmon resonance frequency and the electronic scattering rate

(or the inverse electron mean-free-path), respectively. αpcan be expressed upon combining equation

2.18 and equation 2.19 as

αp=R3εbω2−ω2

p+iωγεb

(εb+3)ω2−ω2

p+iωγ (εb+3).(2.20)

Equation 2.20 will reach a maximum for

ω=ωr=ωp

√εb+3(2.21)

with a half width given by

2. Materials and Methods

γ(εb+3).(2.22)

From that crude model few properties of surface enhancement can be derived. First, γis the

electronic scattering rate, which is a competing process to resonance absorption. If γis high, then the

scattering on the electrons will dominate and thus SE is reduced. Moreover, since γis inversely pro-

portional to the DC conductivity, metals with high internal resistance will therefore exhibit reduced

surface enhancement compared to metal with high conduction properties. For the contribution of

interband transitions to the dielectric properties, it is found that if εbis high, then εis also high,

which will decrease αand therefore the SE. Additionally, a high contribution of interband transitions

will according to equation 2.21 shift the resonance frequency to higher wavelengths.

The effect of particle size on the SE is given by two main factors. The smallest dimension of a

particle has to be at least in the order of the size of a molecule. For too small metallic objects, that is

when they are much smaller than the electron free-mean-path, conductivity will drop and hence SE

will decrease as well. Moreover, if the particle consists of only few atoms, then classical description

as given above may no longer be valid and quantum theory has to be applied to evaluate its electronic

properties. On the other hand, the upper size limit is given by the excitation wavelength. If the object

is much larger than 100 nm, then the incident radiation will excite multipoles instead of dipoles

exclusively. Since multipoles are non-radiative, they cannot contribute to the surface enhancement.

Therefore, the optimal size for a metal particle (or other structures) to show surface enhancement

ranges for coinage metals from 10 nm to 100 nm [20, 29, 30].

In summary, the magnitude of enhancement depends largely on the morphology and the electronic

properties of the metal. The metals that are expected to display the strongest enhancements are

(sorted in decreasing order of provided SE) silver, the alkali metals followed by gold, copper and

the good conductors aluminum, indium, platinum and finally the less good conductors and other

transition metals.

A SER spectrum of a probe molecule on a SE active support differs in many aspects from a normal

Raman spectrum of the very same probe in solution as shown for mercaptobenzene in figure 2.2.

Usually, the overall SER signal intensity ISERS is, given a proper choice of excitation wavelength and

respective support, much stronger than respective Raman signal intensity IRaman. To evaluate now

the intensity gain through surface enhancement, it is necessary to define a few values. Assuming

that Etot denotes the averaged total field strength induced by plasmon excitation, then gwill be the

enhancement factor given by Etot =g E0, where E0is the field strength of the incoming light. The

Raman scattered light will then have the field strength Esca (ωs)∝αEtot ∝αSER g E0(ω1), where

αSER denotes the polarizability tensor of the surface attached molecule. Also, the scattered light is -

given that its frequency ωs=ω1−ωfi can excite surface plasmons - enhanced by the factor g0. The

field strength of a SER scattered radiation is therefore given by

ESERS ∝αSER g g0E0.(2.23)

As shown above, the intensity of a Raman signal is proportional to the square of the induced dipole

moments and hence to the square of the electric field inducing the dipoles and thus equation 2.23 can

be transformed to

ISERS ∝(αSER)2gg02I0.(2.24)

For ωs≈ω1and g≈g0, the electric field will be enhanced by the power of four. The total enhance-

ment G, provided by the metal particle, can then be expressed as

G=

αSER

αgg0

2.(2.25)

2. Materials and Methods

Figure 2.2: Normalized Raman (top) and surface enhanced Raman (bottom) spectra of mercaptoben-

zene obtained with 413 nm excitation. The SE active support was constituted by a rough

Ag surface.

Usually, αSER is equal or similar to αand hence the ratio will be one. However, for molecules

with certain chemical properties, a significant change of the polarizability tensor, leading to high

scattering yields, was observed upon adsorption. Raman signals of these molecules are strongly

enhanced, even though there might be no plasmonic enhancement involved. This effect is called

chemical enhancement effect and is ascribed to interactions between the probe molecule and the

metal surface which in turn change the electronic properties of both [31].

In principle, SER is not only restricted to adsorbed molecules. According to the electromagnetic

theory, the strength (magnitude) of the local electric field decreases with the 1/d3, where dis the

distance to the metal surface [34, 35]. Because the signal enhancement varies with g4, the distance

dependence is accordingly scaled with 1/d12 [35]. For a particle with a diameter of a0, the distance

dependence is described by

GSER (d) = GSER (0)·a0

a0+d12

.(2.26)

GSER (0)denotes the maximum enhancement at the surface. For an a0in the order of a few tenth

of nanometer, the electric field enhancement is drastically reduced at a distance several nanometers

away from the surface.

Surface enhancement cannot only be observed for particle ensembles, but also for bulk materials

that exhibit a nanostructural geometry on the surface. These nanostructures can function as center

for surface enhancement due to the very same mechanism as described for particles, given a proper

size of these structures. In that case, the total field enhancement is a mean value over the complete

surface. The magnitude of maximum and minimum enhancement for a certain nanostructural surface

2. Materials and Methods

geometry can vary strongly as a function of space [20]. Thus, it might be the case that for a metal

structure that exhibits a high mean field enhancement, SER signals of adsorbed molecules may only

arise from a fraction of molecules that sit at so called ’hot spots’. These spots are areas on the surface

showing the strongest field enhancements, which can be several orders of magnitude higher than that

of other areas. In single molecule SER(R)S (vide infra), it is believed that only molecules sitting at

these hot spots will give rise to a spectrum [19]. The determination of the location of these spots

is purely a task of computational methods since no experimental technique exists that can probe the

field enhancement of nanostructures spatially resolved. Since the surface enhancement effects can be

described satisfyingly by classical electromagnetic theory, field enhancement arising from different

nanostructures can be calculated by modeling these structures and solving the related Maxwell equa-

tions [20]. Due to computational restriction, there exists a variety of methods which can simplify the

calculations, allowing reasonable numerical solutions of the Maxwell equations. The most promi-

nent method is the finite difference time domain approach (FDTD) [36, 37]. This method is along

with other approaches grid-based in which the structure is modeled by finite elements. In this way,

nanostructures with dimensions of several hundreds of nanometers can be treated theoretically with-

out facing computational limitations. These kind of calculations are of high potential since they can

either be used to predict which geometry exhibits the highest surface enhancements or may help to

rationalize experimental results [118, 233]. Also, they can be applied to investigate complicated com-

posites including metal/metal combinations and structural geometries which are not yet accessible

by synthesis and thus are not pliable for experiments.

2.0.5 Surface Enhanced Resonance Raman

The surface enhancement can boost the Raman signal intensity drastically, making this method very

sensitive and thus particularly interesting for probing adsorbed molecules. The sensitivity can be fur-

ther improved by combining the surface enhancement with the molecular resonance Raman effect, in

case the probe molecule, e.g. a dye, exhibits an electronic transition close to the wavelength required

for exciting plasmon resonances of an underlying metal support. The resulting technique is called sur-

face enhanced resonance Raman (SERR) spectroscopy and is suitable to probe simple dye molecules

as well as to monitor large molecules such as enzymes that contain one or more chromophores (vide

infra).

The choice of the substrate is crucial when employing SERR spectroscopy. Since most of dyes and

absorbing molecules exhibit strong electronic transitions that lie in the visible region, it is also neces-

sary to have a metal support that can be excited using the very same wavelength. Most of the metals

are therefore not suitable for this purpose, since they either show low intrinsic surface enhancement

or the necessary excitation wavelength is shifted to the UV or infrared region. In this respect, Ag

and Au are often employed since they can be excited using visible light [45], while the exact posi-

tion of the plasmon resonance frequencies depend on the structure and the geometry of the Ag/Au

support (vide supra). Therefore, Ag or Au in form of nanoparticles are often employed in SERR

experiments, either for detecting molecules in solution or coated with good Raman scatterers to be

used as markers in spatial resolved confocal Raman measurements [46–48]. One of the advantages

of nanoparticles is that the exact plasmon absorption wavelength can be tuned by changing shape,

radius or dielectric properties such as coating with an additional metal layer [48–50]. Thus, the plas-

mon resonance frequency can be adjusted to match the electronic absorption frequency of the sample

molecule, which in turn provides high sensitivity and selectivity. Also, solid electrodes made of Ag or

Au are used as SERR supports [35]. To obtain the field enhancement, the surface of these electrodes

has to exhibit a roughness on the nanometer scale. Since the roughness is based on irregular shaped

metal structure geometries, the plasmon resonance frequency of these electrodes is not well-defined

as in the case of monodisperse nanoparticles but is distributed over a region, such that these supports

can be used at different excitation wavelengths. Additionally, these electrodes can be employed in

2.1 Properties of Electrochemical Interfaces

electrochemical experiments, enabling the coupling of spectroscopy and electrochemistry to vibra-

tional spectro-electrochemistry [35]. This is particularly interesting for probing redox enzymes and

electro-catalytic reactions [38, 51–54]. Most enzymes carry a chromophore unit that constitutes also

the active site of the catalyzed reaction. If the plasmon excitation wavelength is tuned to match the

respective electronic transition of the chromophore, vibrational bands originating from the chro-

mophore unit will be selectively enhanced. Hence, SERR spectroscopy provides also a powerful tool

to investigate structure and mechanism of enzymes without interfering contributions from the protein

matrix [35, 38, 39, 79]. Vibrational bands of the protein backbone can be obtained by performing IR

absorption spectroscopy or surface enhanced infrared absorption (SEIRA) spectroscopy [35, 40].

2.1 Properties of Electrochemical Interfaces

The insertion of a bare metal electrode into an aqueous solution leads to adsorption of ions on the

electrode surface and thus to the formation of an electrical double layer at the solid/water interface as

shown in figure 2.3. The distrubtion of the potential Φat such an interface can be described using the

Gouy-Chapmann-Stern theory. According to this, the first layer of adsorbed ions is called the Stern

layer and its properties depend on the kind of metal and the type of electrolyte. The electrode together

with the layer of adsorbed ions is treated as a molecular capacitor, and thus the potential drops linearly

from the electrode surface to dc, which denotes the thickness of the Stern layer. Beyond the Stern

layer, the distribution of ions is diffuse, and thus the potential drops exponentially with increasing

distance from the metal surface.

The charge densities at the interface can be quantified using the approach of Smith and White [55].

Accordingly, the charge density on the metal is given by

σM=ε0εC

(ΦM−ΦC),(2.27)

where ε0is the permittivity and dCand εCrepresent the thickness and dielectric constant of the

Stern layer, respectively. ΦMand ΦCdenote the potential of the metal and the potential measured

after the Stern layer as shown in figure 2.3, respectively. The charge density in the solution can be

derived from the Gouy-Chapman distribution in which an exponential decrease of the potential with

increasing distance from the Stern layer is assumed. The decrease is scaled with the potential differ-

ence between ΦCand ΦS, the solution’s potential. Accordingly, the charge density can be expressed

σS=−ε0εSκ2kT

esinhe

2kT (ΦC−ΦS).(2.28)

Here, κdenotes the inverse Debye length while kand Thave the usual meaning. The charge

densities vary upon applying an electrode potential, i.e. changing the value of ΦM. Two special cases

can be distinguished. If ΦMis equal to ΦS, then σMwill become zero. This value for ΦMis called

the potential of zero charge Epzc and is roughly - 0.7 V and + 0.2 V vs SHE for the metals Ag and

Au, respectively. If ΦMis very high with respect to ΦC(or ΦS), then the consequence will be a sharp

potential drop along the Stern layer. This fast potential drop causes an high electric field EFthat is

determined by

EF=φM−φC

=σM

ε0εC

.(2.29)

The field strength depends also on the kind of bound anions since these ions determine the prop-

erties of the Stern layer. For sulfate as binding ion, the electric field can reach magnitudes up to 109

V m−1at an applied electrode potential of + 0.2 V [35].

2. Materials and Methods

Figure 2.3: Schematic representation of an electrical double layer and the potential distribution at the

solid/water interface. The first layer of adsorbed ions is called the Stern layer, which has a

thickness of dc−d0. The potential Φdrops linearly with increasing distance from d0to dc.

Beyond the Stern layer, the distrubtion of ions is diffuse. Accordingly, the potential Φ(d)

more far away from the electrode can be approximated using an exponential decay.

2.1.1 Self Assembled Monolayer - SAM

Adsorption of biomolecules on bare metal surfaces may cause denaturation due to the high electric

fields present at the electrode/water interface [56]. To avoid such effects, the metal surface is usually

covered by bio-compatible coatings [57–59, 67]. The most versatile coating type is based on the

self-assembly of amphiphilic alkanethiols of the type R-(CH)n-SH, where ndenotes the length of

the alkyl chain. The sulfur group can bind strongly to a variety of metals, forming a monolayer

via self-assembly (self assembled monolayer, SAM) [60, 61]. The properties, such as density of

package, degree of order or metal-S-chain angle, of the resulting monolayer depend on the type of

the employed SAM, i.e. on the number of nand the nature of R, which represents a functional head

group. Depending on the properties of the protein, R can be varied to ensure an efficient adsorption.

For example, the heme protein cytochrome ccan be effectively adsorbed using negatively charged

SAMs (R is for example a carboxylic acid group) via electrostatic forces. Figure 2.4 illustrates the

different mechanisms for protein adsorption using different kind of head groups. The most complex

immobilization strategy refers to the cases B and D where adsorption of the protein is achieved via

formation of covalent bonds between the SAM and the molecule [38, 157].

The choice of R also affects the charge density at the interface. In case of R = CH3, the smallest

charge densities were reported while for phosphate, sulfate and polyanions the highest charge den-

sities and therefore the highest electric fields were measured [35]. Also, the charge on the electrode

affects the chemical properties of the head groups. For example, adsorption of carboxyl terminated

SAMs on an Ag electrode will increase the pKaof the carboxylate group and thus in turn increase

the electric field strength. This can be further manipulated by adjusting length, i.e. the effective

2.2 Heme proteins

Figure 2.4: Different modes of protein (black polygon) immobilization. The stabilizing interaction can

be mediated via electrostatic forces (A), covalent bonds (B) or hydrophobic forces (C).

Additionally, the protein can also be immobilized via typical protein tags as used for HPLC

(D).

separation between the headgroup and the electrode surface, whereas small nlead to high electric

fields and vice versa. For n=11, the electric field strength was measured to be around of 109V/m

[62–64]. This is in the order of values expected for phospholipid membranes [65]. A more profound

introduction to SAMs can be found in the cited literature [66, 67].

2.2 Heme proteins

Heme proteins carry an iron porphyrin cofactor. These proteins play a key role in many reactions

in biological systems and can act as electron and oxygen carriers, signal transducers and function

as metabolizing enzymes. The heme groups can be distinguished by the chemical constitution of

the porphyrin macrocyle and the ligation, spin and oxidation state of the heme iron center atom.

The chemical formula for a-, b- and c-type hemes are shown in figure 2.5. Type-bhemes carry two

vinyl substituents whereas type-ahemes exhibit a vinyl and a formyl functional group. Additionally,

type-ahemes possess an aliphatic substituent whose length can differ from organism to organism.

Both heme types are linked to the rest of the protein by non-covalent interactions. For example, the

binding to the protein matrix can be achieved by the coordinating axial ligand of the iron atom. In

contrast, type-chemes exhibit two vinyl groups that are used to link the cofactor covalently to the

protein matrix by forming thioether bridges with cysteine residues. Furthermore, within one heme

type, the coordination pattern of the heme iron can differ depending on the exogenous or endogenous

axial ligands. Either both axial positions are occupied or one position remains vacant, according to a

sixfold (6c) or fivefold (5c) coordination pattern, respectively. 5c and 6c coordination with low field

ligands lead to a formation of a complex where the iron atom is in a high spin state (HS), in analogy

2. Materials and Methods

Figure 2.5: Chemical constitution of the different natural appearing heme types a,band c. All hemes

consist of a conjugated tetrapyrrol macrocyle coordinating a metal atom. The peripheral

substituents attached to this macrocyle differ depending on the type of heme.

to octaedrical complexes where the five d-orbitals of the iron atom split up into three degenerate eg

and two lower energetic degenerate t2gorbital. High field ligands, in contrast, favor the formation of

a low spin iron state (LS). Beside this, the iron atom can vary between a ferric and a ferrous oxidation

state in which the formal charge on the iron atom is 3+and 2+, respectively. The transition between

the various spin, coordination (or conformational) and oxidation states depends on many factors and

can be probed with different spectroscopic, electrochemical and spectro-electrochemical techniques.

2.2.1 Cytochromes

Cytochromes are heme proteins, which function as electron carriers (e.g. cytochrome c) or enzymes

(e.g. cytochrome P450) [68]. They are widely spread throughout animal and plant life and appear

in the different types a,b,cand ddepending on the chemical constitution of the heme group (vida

infra) [69].

Cytochrome bis an integral membrane protein with a molecular mass of about 40 kDa. Similar to

the other cytochromes, there exists a variety of modifications of that protein type depending on the

organism the enzyme was isolated from. The archetype is the cytochrome bfrom the cytochrome

bc1 complex, i.e. the cytochrome creductase (complex III of the mitochondrial respiratory chain of

eukaryotes). This cytochrome contains two low spin heme groups, which can be distinguished by their

electronic absorption and their redox potentials. Both iron atoms are ligated by two highly conserved

histidine axial ligands. The major protagonist of a-type cytochromes is the cytochrome complex aa3.

This membrane bound complex is a subunit of the cytochrome coxidase enzyme (complex IV of the

eukaryotic respiratory chain), which supplies the electrons for the reduction of oxygen to water, to

generate a proton gradient for ATP synthesis. Cytochrome aa3contains two a-type heme groups, one

in the low spin (a) the other one in the high spin configuration (a3) [70, 71]. It has, additionally, two

copper atoms and catalyzes the oxidation of mitochondrial cytochrome c, a small soluble enzyme

with 103 to 112 amino acid residues and a molecular mass of approximately 12 kDa.

Under normal conditions, cytochrome cis attached to the inner mitochondrial membrane where it

functions as an electron carrier and transports electrons from complex III to complex IV, promoting

the oxidative phosphorylation. As shown in figure 2.6, it accommodates one low spin heme group that

2.2 Heme proteins

Figure 2.6: Left. 3D structure of cytochrome c. The covalently attached c-type heme is highlighted.

Right. 3D structure of the heme group with the axial ligands histidine (top) and methionine

(bottom).

is axially coordinated by a methionine and a histidine ligand. The redox potential of the Fe2+/Fe3+

transition is comparably high with around 270 mV vs. SHE. Furthermore, it possesses a relatively

high dipole moment of roughly 300 Debye, which alters only slightly upon reduction [128, 129].

Additionally, cytochrome cexhibits a partially positively charged area that can be used for electro-

statically mediated immobilization. Conformational transitions are reported upon high pHs (alkaline

transition), under oxidative stress and at high electric fields and is related to an axial re-ligation

[38, 93, 98, 99]. The loss of the axial ligand methionine, forming a 5cHS species, is accompanied

by a lowering of the redox potential by several hundreds of mV [38]. The coordination site can be

occupied by other ligands such as a lysine or transiently by another histidine, both yielding a 6cLS

heme species. Also, the site can remain vacant which is reported to lead to an increase of peroxidase

activity of cytochrome c[170]. Furthermore, the transition to a 5cHS iron is believed to be of partic-

ular importance for altering the function of cytochrome cas an electron carrier to a signal transducer

in the apoptotic reaction cascade [161].

Finally, cytochrome dare proteins which contain a heme, where the iron atom is chelated by a

tetrapyrrol marcocyle in which the degree of conjugation of double bonds is lower than in a normal

porphyrin. Specifically, this is the case for dihydroporphyrin (chlorin, reduced by one double bond),

tetrahydroporphyrin (reduced by two double bonds) and siroheme. Heme dis formerly denoted as

heme a2.

2.2.2 Resonance Raman Spectroscopy of Cytochromes

Heme proteins have been intensively studied over the last decades using various spectroscopic

techniques. The heme group constitutes a chromophore unit that gives rise to an intensive UV/Vis

2. Materials and Methods

Figure 2.7: UV/Vis optical spectrum of oxidized (red) and reduced (blue) cytochrome c. In RR experi-

ments, a laser excitation line at 413 nm (purple) is used to obtain strong resonantly scattered

signals. Figure taken from [76].

absorption spectrum. This spectrum exhibits typical features that can be rationalized using molec-

ular orbital (MO) theory, as proposed by Goutermann et al [72]. In this model, a D4hsymmetry

is assumed for the porphyrin ring, which is treated as a two dimensional system of conjugated π

bonds. Accordingly, the highest occupied MOs have a1uand a2usymmetry and are similar in energy

[73, 74]. The transitions occur into the lowest unoccupied MO, which has egsymmetry and is

double degenerated. The transition from the a2ugive rise to a very intense band (γor Soret Band)

at roughly 400 nm while the transition originating from a1uleads to the lower energy bands in the

region around 540 nm (Q or αand βband), which is split due to a (0,0)and a (0,1)vibrational

transition, respectively. Both electronic transitions are of Eusymmetry and thus are strongly mixed

by configuration interaction such that the transition dipole moments of both transitions combine

additively and substractively with the γor Soret band gaining most of the intensity. A typical UV/Vis

spectrum of cytochrome cis depicted in figure 2.8.

Therefore, in order to obtain high quality resonance Raman spectra of heme proteins, the exciting

laser line has to be chosen to match either the γor the αor βelectronic transition. Assuming a D4h

symmetry for the porphyrin marcocycle, the transition dipole moment vector lies for both transitions

in the x,yplane, i.e. in the porphyrin plane, hence resonance enhancements are only expected for

the in-plane modes with A1g, B1g, A2gand B2gsymmetry. Upon Soret excitation, the RR spectrum

is dominated by the total symmetric A1gmodes according to the Aterm resonance enhancement.

The high intensities of these modes are mainly due to the large oscillator strength of the electronic

transition. Bterm enhancement plays only a minor role for these modes due to small excited state

displacements. In contrast, the non-total symmetric B1g, A2gand B2gmodes are enhanced strongly

by the vibronic coupling (Bterm) and gain intensity under Q band excitation. Under these conditions

B1gmodes are mainly enhanced. Also, modes with A2gsymmetry are observed, which are Raman

inactive under non-resonant excitation conditions. However, the symmetry of natural porphyrin

macrocycles is lower than D4hsince the individual side chains lead to an asymmetric substitution

2.2 Heme proteins

pattern. This has some consequences for the resonance Raman activity of the different vibrational

modes [77–79]. For example, the vinyl and formyl substituents present in aand btype hemes can

couple with the conjugated π-electron system of the macrocycle and thus the internal modes of

these side chains are also enhanced. For ctype hemes the vibrational modes of the thioether bridges

are coupled to the porphyrin ring, leading to a prominent resonance enhanced band with high C-S

stretching contributions at around 700 cm−1[80]. Additionally, also out-of-plane modes can gain

resonance enhancement due to coupling of in-plane and out-of-plane modes and thus ’borrowing’

intensity via respective Aor Bterm enhancement (vide infra).

In figure 2.8, Soret and Q band excitation spectra of ferrous and ferric cytochrome care shown.

Due to a large number of experimental data available, the vibrational bands are well assigned [81–86].

The region between 1200 cm−1and 1700 cm−1is the so-called marker band region. The band pattern

there reflects the oxidation, spin and ligation state of the heme iron atom. Under Soret excitation,

the most prominent band in the ferric spectrum is the ν4(A1g) mode at around 1371 cm−1which

corresponds to an almost pure C-N stretching mode. Other important modes are the ν3(A1g) and

the ν10 (B1g) mode. Latter one dominates the spectrum upon Q band excitation. Upon reduction, all

these bands shift to lower wavenumbers. The ν4mode displays the largest shift by roughly 10 cm−1

to ca. 1361 cm−1. This can be rationalized by taken into account the electronic interplay between the

iron and the porphyrin. The additional charge on the iron atom increases the back-bonding properties

and thus increases the electron density in the π∗orbitals of the macrocycle. This in turn weakens the

C-N bond strength which finally leads to the downshift. Coordination with other electron-rich axial

ligands such as thiolate can even cause shifts up to 25 - 30 cm−1, as found for cytochrome P450

[79, 87, 88].

The spectral region below 1000 cm−1displays bands associated mainly with protein-heme inter-

actions due to the covalent attachment of the heme group to the protein backbone. The low frequency

region between 300 and 450 cm−1is the so-called fingerprint region and contains spectral signatures

of modes arising from bending and out-of-plane modes of substituents and the porphyrin macrocy-

cle. This region is unique for cytochrome c. The band intensities and frequencies are indicative of

transformation processes associated with the heme group. Cytochrome cexhibits a variety of confor-

mational states which were addressed by many electrochemical and spectroscopic studies [89–94].

Most of these conformational transitions are restricted to oxidized cytochrome cexclusively due to a

weak Fe-S(Met80) bond in this redox state. As aforementioned, this bond can be easily broken and

the vacant axial position can be occupied by another amino acid residue. This re-ligation is, however,

also associated with movements of protein peptide segments. All these changes are nicely reflected

in the marker band and the fingerprint region of a resonance Raman spectrum. For example, upon

lowering the pH a bis-histidine (His-33 or His-26/His-18) ligated 6cLS heme species can be formed

[95]. The attachment of this high-field ligand leads to an increase of the iron-porphyrin bond strength

accompanied by a smaller core size and hence an upshift of the marker bands, in particular of the ν4

(to ca. 1374-1375 cm−1), ν3(to ca. 1505-1507 cm−1) and ν10 (to ca. 1639-1644 cm−1). This ligation

pattern can be distinguished from the likewise 6cLS conformation that is formed upon alkaline pH,

known as the alkaline transition of cytochrome c[96, 97]. There, a Lys(72,73 or 79)/His(18) ligation

configuration is present [98, 99]. The different ligation requires a different tilting of involved peptide

segments, which has impact, in particular, on the low frequency region of the RR spectra. Based on

that, a spectroscopic distinguishing between both states is possible [93]. Beside the 6cLS configu-

ration, one may also find two HS states in which the iron atom is either five- or six-coordinated. In

the 5c and 6c states the former axial position of Met80 remains vacant and is occupied by a water

molecule, respectively. This leads to a significant down shift of the ν3band to either 1488-92 cm−1or

1475-80 cm−1, respectively. It is known that all these states are involved during the folding processes

of cytochrome c, but are also formed upon interaction between cytochrome cand highly charged

surfaces such as metal electrodes, phospholipid vesicles or SDS micelles [90, 91].

2. Materials and Methods

Figure 2.8: Resonance Raman spectra of reduced (top, bottom) and oxidized (middle) cytochrome c

obtained with 413 nm and 514 nm excitation, respectively. The significant marker bands

ν4,ν3and ν10 are highlighted to track their frequency and intensity changes upon redox

transition.

2.3 Electron Transfer Theory

Electron transfer (ET) reactions play a fundamental role in chemical and biochemical processes. To

understand these processes, it is crucial to have profound knowledge about the properties that control

these transfer reactions. The first generally accepted theoretical description of electron transfer was

provided by R. A. Marcus [100–104].

2.3.1 Homogeneous ET and Classical Marcus Theory

The Marcus theory is usually used for outer-sphere ET reactions between a donor (D) and an acceptor

(A) molecule. The process of ET can be divided in three steps. First, the diffusion of Dand Ato form

a complex takes place. The second step is the actual ET process: D/AkET

−−−*

)−−−

k−ET

D+/A−. In the third

step, Dand Afinally seperate by diffusion. The Gibbs energy of such an ET reaction is given by

∆G0=e(E0

D−E0

A+wP−wR),(2.30)

where E0

Dand E0

Aare the redox potentials of the redox couple D/D+and A/A−, respectively. wP

and wRdenote the required energy to form D/A(reactant) and to induce the separation of D+/A−

(product), respectively. The potential energy of these states, i.e. of the reactant and the product, is a

2.3 Electron Transfer Theory

Figure 2.9: Gibbs energy curves as a function of the reaction coordinate for the reactant (R) and product

(P) state. ∆Gis defined as the Gibbs energy difference between the parabola minimum of R

and P. ∆Gεdenotes the necessary activation Gibbs energy and is defined as the difference

of the Gibbs energy minimum of R and the Gibbs energy at the intersection between both

parabola. The reorganization energy λis the energy necessary to distort the geometry of

the reactant state to match with the geometry of the product state at minimum energy.

complicated multi-dimensional energy surface including also the potential energy of the surrounding

solvent sphere. For simplification and in analogy to transition state theory, a reaction coordinate is

introduced to reduce the problem to one dimension. Additionally, it is more convenient to plot free

Gibbs energies instead of potential energies, since they can be approximated using simple parabola.

Such an exemplary plot is shown in figure 2.9. The reacant (R) and the product (P) are represented

by the same parabola but with different vertices. This is justified for small changes (both in ener-

gies and internal coordinates) of the complexes upon ET reaction. To induce an electron transfer,

the reactant system has to reach the intersection of both parabola. This can be achieved classically

for example by thermal activation. According to classical transition state theory, the first-order rate

constant of such an ET process is given by

kET =κelνnexp−∆Gε

kBT.(2.31)

κel and νndenote the electronic transmission coefficient and the frequency of the system reaching

the transition state (≈1013s−1), respectively. ∆Gεrepresents the Gibbs energy of the activation as

shown in figure 2.9. Since ∆Gεis not accessible by experiment, it is more convenient to express the

equation 2.31 as a function of ∆G0and λ, which represent the standard Gibbs energy of the reaction

at a certain donor/acceptor distance rDA and the reorganization energy, respectively. Using analytical

geometry, ∆Gεcan be expressed as

2. Materials and Methods

∆Gε=λ+∆G02

4λ.(2.32)

The reorganization energy λis also shown in figure 2.9 and is defined by the energy required

to bring the reactants to the same nuclei configuration where the products exhibit minimum energy

without transferring an electron. λconsists of two additive terms: λin and λout. The first term repre-

sents the solvent-independent inner term which arises from structural differences between the equi-

librium configurations of the reactants and products. The second term contains the solvent reorgani-

zation energies and accounts for differences in orientation and polarization of the surrounding solvent

molecules of the reactant and product complex. Both contributions to the reorganization energy can

be calculated theoretically if a proper model of the transition state is available. Upon combining these

substitutions, one yields the classical Marcus equation

kET =κelνnexp −(λ+∆G0)2

4λkBT!.(2.33)

As can be derived from equation 2.33, kET increases for decreasing ∆G0and reaches its maximum

at −∆G0=λ(∆Gε=0). A further decrease of ∆G0does not increase the reaction rate, but it leads

to a decrease of kET for these kind of highly exergonic reactions due to a re-increase of ∆Gε. The

region where ∆G0>λis called the Marcus inverted region and was confirmed by experiments

[105, 106]. Its prediction is one of the advantages of the Marcus theory in comparison to other ET

models [107].

The classical Marcus theory can predict very well rate constants of adiabatic (κel 1)ET reactions

but fails with respect to non-adiabatic (κel << 1)processes. In these reactions, tunneling of elec-

trons from Dto Aand nuclei from reactant to product states play a major role and thus a quantum

mechanical approach has to be chosen [108–111]. The first major difference is the introduction of the

electronic coupling element ˆ

Hel, defined as the overlap of the electronic wavefunction of the donor

and acceptor ψDand ψA, respectively.

HDA =ψ0

Rˆ

Helψ0

P(2.34)

Hel denotes the electronic Hamilton operator in the Born-Oppenheimer approximation. The mag-

nitude of the coupling decays exponentially with separation of donor and acceptor raccording to

|HDA(r)|2=|H0|2exp(−β(r−r0)).(2.35)

H0denotes the Hamilton operator at the Van-der-Waals distance r0.βrepresents the tunneling

parameter and depends on the separating medium. The quantum mechanical approach treats the reac-

tants and product as complete system of states represented by nuclear and electronic wave functions.

To estimate the transition probability from one state to the other, time dependent perturbation theory

has to be applied. In analogy to Fermi’s golden rule, one obtains for the transition probability ωj

from the reactant (R) to a product state (P)

ωj=2π

hH2

RP ∑

i,jχ0

Pi|χ0

R j2δi j (εPi −εR j).(2.36)

Dχ0

Pi|χ0

R jEdenotes the Franck-Condon (FC) integral containing χ0

Pi and χ0

R j, the nuclear wave

function of the product and reactant in the state jand iwith the respective energy εR j and εPi,

respectively. The transition probability is summed over all possible states i.δdenotes the Dirac

delta-function which ensures energy conservation (transition from state ican only lead to product

2.3 Electron Transfer Theory

state jwhich has the same energy). Accordingly, the first-order rate constant of an ET process can

be derived by multiplying the probability with the number of possible states and is expressed as

kET =2π

h|HDA|2FCWD.(2.37)

FCWD denotes thereby the Franck-Condon-weighted-density-of-states term in which the FC inte-

grals are weighted according to the probability of occupation P(εR j)of respective states using the

Boltzmann distribution statistic. FCW D has the form

FCWD =∑

i,jχ0

Pi|χ0

R j2P(εR j)δ(εPi −εR j).(2.38)

In a fully quantum mechanical description, the sum in equation 2.37 is taken over all vibrational

modes including those of the surrounding solvent molecules. However, since these modes have lower

frequencies than the internal molecule vibrations, the solvent molecules are often treated classically.

The refined resulting equation is called the semi-classical Marcus equation and has the form

kET =2π

h|HDA|21

√4πλokBT×

∑

i,jχ0

Pi|χ0

R j2P(εR j)exp"−∆G0+εPi −εR j +λo2

4λokBT#.(2.39)

In the high-temperature approximation, the equation further reduces to

kET =2π

h|HDA|21

√4πλ0kBTexp−(λ+δG0)2

4λkbT(2.40)

2.3.2 Heterogeneous ET

In heterogeneous ET reactions, one of the reaction partners is replaced by an electrode. The driving

force of these heterogeneous reactions is given by the applied overpotential η.

η=E−E0(2.41)

Edenotes the applied electrode potential and E0represents the redox potential of the surface ET

reaction defined by the Nernst equation

E=E0−kBT

ne ln(cred

cox

).(2.42)

cred and cox denote the respective concentrations of the electrochemically active species while n

represents the number of transferred electrons per reaction formula.

In analogy to homogeneous reactions, one finally obtains the expression for non-adiabatic ET reac-

tions (Marcus-DOS,density of states) [112]

kET =2π

√4πλ0kBTZdεf(ε)ρ(ε)|HDA(ε)|2P(εRj)exp−(λ+δG0(ε,η)2

4λkbT.(2.43)

Here, the acceptor or donor is replaced by the metal electrode. The sum is accordingly made over

all possible energy states εof the metal and therefore replaced by the integral and extended with the

2. Materials and Methods

state density function ρ(ε). Furthermore, ρ(ε)|HDA(ε)|2=|V(ε)|2yields the electronic coupling

strength between the electrochemical species and the electrode. The expression contains, in analogy

to the Boltzmann distribution, the Fermi distribution function f(ε), which accounts for the different

occupation probability of different energy states εof the metal.

The equation can be further simplified by introducing few assumptions [113–115]. For the oxidation

one yields

kox =ApπλkbT

e1+er f eη−λ

2√λkBT(2.44)

and accordingly for the reduction

kox =ApπλkbT

e1−er f eη+λ

2√λkBT.(2.45)

The error function er f is defined as

er f (x) = 2

√πZx

exp(−t2)dt (2.46)

Adenotes a parameter that depends on |HDA(ε)|2and r. Furthermore, the Fermi-Dirac distribution

function was replaced by a ladder function accounting for the difference in energies. Also, contribu-

tions from energy levels far away from the Fermi level are not considered.

3 Experimental Details and Instrumentation

This chapter contains details on the methods and equipment of the experiments presented in the

attached publications.

3.1 Confocal Raman Spectrometer

SE(R)R and RR spectra were collected using a LabRam HR-800 (Dylor-Jobin Yvon-Spex) confo-

cal Raman microscope (Olympus Bx 40) equipped with a nitrogen cooled CCD detector. The 413

nm and the 514 nm line of a Krypton (cw Coherent Innova 330c) and an Argon ion (cw Coherent

Innova 70c) laser were used as excitation lines. The laser light was focused onto the sample using a

Nikon 20xobjective with a numerical aperture N.A. of 0.35 and a working distance of 20.5 mm. The

Raman signals were collected in 180◦back scattering geometry. A schematic representation of the

experimental Raman setup is shown in figure 3.1.

After leaving the laser tube, the light passes two electro-optical laser intensity modulators (Pockel

cells, Linos M 0202). With these modulators, laser pulses in the ms-time scale are created, enabling

time resolved measurements (vide infra). Behind the spectrometer entrance, an interference filter

(IF) is placed to remove laser plasma lines. The laser light is then focused through a pinhole onto

a notch filter (NF) that reflects the beam into the microscope exit. The laser light is focused onto

the sample using a microscope objective. The scattered laser light is collected through the same

objective in back-scattering mode. The scattered beam passes the notch filter again, which is now

transparent for the frequency shifted light. Behind the notch filter, the light beam is reflected by a

mirror on the confocal hole (1000 µm) and is subsequently focused on the slit entrance (100 µm)

of the monochromator unit. The monochromator is setup in the Czerny-Turner mode and can be

operated with two different gratings, 2400 mm−1and 514 mm−1, respectively. The dispersed light

is finally focused on a nitrogen cooled CCD chip. This chip consists of 2048×512 single pixels

which can be used for light detection. In most of the experiments, a binning factor of two was used.

This yields a resolution of 1 cm−1with an increment per data point of 0.28 cm−1and 0.15 cm−1

upon using the 413 nm and the 514 nm excitation line, respectively. The spectrometer is controlled

using the company supplied software LabSpec v4.07 (Jobin Yvon, 2007). For calibration, the intense

mercury line at 435.8238 nm and at 546.074 nm was used, respectively. The accuracy of the band

positions was ±0.5 cm−1.

3.2 Electrochemical Measurements

Electrochemical measurements and electrode roughening were carried out with a computer con-

trolled multistat (CH Instruments 660b, Austin, TX, USA). The control interface was supplied by the

company software CHI660c v6.13, which allowed also data evaluation (baseline cut, determination

of peak positions and widths and peak integration). The electrochemical cell was a homemade three

electrodes containing electrochemical cell with a volume of ca. 5 ml. As counter electrode, a thick

platinum metal sheet was used. An Ag/AgCl 3 M KCl (DriRef, World Precision Instruments) served

as reference electrode. Ag ring electrodes were fabricated from Ag rods (99.9 %, Good Fellow

3. Experimental Details and Instrumentation

Figure 3.1: Schematic representation of the Raman setup. Details are given in the text. CCD charged

coupled display; H hole; L lens; M mirror; NF notch filter; P pinhole; S slit.

GmbH) and exhibited an averaged geometrical area of around 1 cm2.

3.3 Resonance Raman and Surface Enhanced Resonance Raman

Measurements

Resonance Raman measurements were carried out in a cylindrical quartz cuvette (Hellma). Sample

volumes ranged from 50 to 500 µl. During the measurement, the cuvette was rotated using a home-

made motor device. The applied laser power was 1 to 5 mW. Spectral acquisition times varied from

5s to 60s depending on the desired spectrum quality.

SE(R)R measurements were carried out using a cylindrical Ag bulk ring electrode as SE active mate-

rial. For that purpose, the Ag ring electrode was polished with different sand papers with increasing

order of grain size. In total, the polishing procedure consisted of five steps (grain sizes 1000, 20, 9,

5, 0.3 mic). After each step, the Ag electrode was sonicated for five minutes in water and in ethanol.

To obtain roughnesses on the nanometer scale, the blank polished electrode was finally roughened

electrochemically. Therefore, the polished Ag ring was mounted into a three electrode containing

electrochemical cell filled with 0.1 M KCl electrolyte solution. Potentials jumps from 0.5 to - 0.5

V were applied, and the Ag electrode oxidized and reduced, respectively. Details can be found in a

protocol published elsewhere [116, 117]. Due to the occurring nucleation reactions associated with

3.3 Resonance Raman and Surface Enhanced Resonance Raman Measurements

Figure 3.2: Left. SEM picture of an electrochemically roughened Ag electrode surface. Right.

Schematic representation of the electrochemical cell for SERR measurements. The rough-

ened Ag electrode is mounted on a rotating holder and functions as working electrode

(WE). CE counter electrode. RE reference electrode. Details are given in the text.

the re-reduction, a nanometer scale coral like surface structure of the Ag electrode was obtained.

Figure 3.2 shows a SEM picture of an Ag electrode surface after electrochemical roughening. This

kind of electrodes functioned as the basis for all the SE(R)R measurements.

To generate hybrid devices, the electrode was subsequently coated with silica layers of differ-

ent types and subsequently charged positively by attaching a silica compound which exhibited an

amino-terminated headgroup. This positively charged surface can be used for immobilization of

MeM+ClnM−n(Me = metal) ions for subsequent metallation by reduction. The so fabricated hybrid

electrode will be introduced in the Results section [118].

For protein studies, the electrode was coated with a proper SAM for protein immobilization. In most

cases, carboxyl-terminated alkanethiol compounds of variable chain length were used (see section

Self Assembled Monolayer). The formation of a monolayer was achieved by incubating the freshly

roughened electrode into a 1 mM ethanolic solution of alkanethiols over night. The quality of the

formed SAM was checked by performing cyclic voltammetry [119].

The SE active Ag electrodes were mounted on a homemade electrode holder system, which exhib-

ited a sliding contact between electrode and holder. By connection to an external motor, it was

possible to constantly rotate the Ag rings during measurements. This was necessary to avoid

laser induced sample degradations when working with biological samples. Moreover, the rotation

ensured an accumulation of signals arising from many different surface features. To complete the

spectro-electrochemical device, an adapter cell made of plastic (Delrin) was finally attached to the

holder, compassing the working electrode. The adapter cell contained ca. 10 mL of an oxygen-free

buffer solution and a platinum wire that acted as counter electrode. An Ag/AgCl 3 M KCl (DriRef,

World Precision Instruments) was placed into the cell via a special entrance and functioned as

reference electrode. Furthermore, the cell exhibited an optical window of ca. 40 mm in diameter

for entrance and exit of laser and scattered light, respectively. A schematic representation of the

spectro-electrochemical cell is shown in figure 3.2. In all SE(R)R experiments, potentials were

adjusted using a potentiostat (potentiostat/galvanostat model 263 A, EG&G Princeton Applied

Research). Potential dependent (stationary) measurements were carried out by applying potentials

to the working electrode. The system was led to equilibrate for 30 s to 1 min before a spectrum was

acquired.

3. Experimental Details and Instrumentation

Figure 3.3: Schematic representation of the time resolved SERR measurement for one delay time δ.

Ciand Cfdenote the concentration Cof the surface adsorbed molecules (in blue) at the

initial potential Eiand at the final potential Ef(in black), respectively. PS denotes the

pulse time, i.e. the time Efis applied to the working electrode. RT denotes the recovery

time, i.e. the time Eiis applied after the potential jump in order to recover initial conditions.

A description of the sequence of measurement is given in the text.

Time resolved experiments were carried out by coupling potential jumps with short measure inter-

vals [38]. For that purpose, a multi-channel delay generator was used to connect the potentiostat

and the laser intensity modulators, which allowed defined spectra acquisition after different reaction

times. The measurements itself followed a scheme as summarized in figure 3.3. First, a potential

jump from an initial potential Eito a final potential Efwas triggered at the working electrode. After

a defined delay time δ, the measurement started by setting the modulator on transmisson for a certain

period of time (measure interval) ∆. Then, the potential was set back to Eito let the system recover

to initial conditions for another period of time RT. Since ∆is usually very short, this procedure had

to be repeated for one δmany times until the resulting sum spectrum was of acceptable quality. By

varying δ, the complete relaxation process, triggered by the applied potential jump, could be probed

SERR spectroscopically. The precise values for δ,∆and RT depended on the investigated reaction.

Normally, δand ∆is substituted by δ0, which is calculated via δ0=δ+∆

4 Results

This chapter summarizes the results obtained in the particular projects of this thesis. Each topic is

introduced through a short description of the broader context. The publications are attached in the

bibliography.

4.1 Electron Transfer Properties of Cytochrome cat Electrochemical

Interfaces

Cytochrome c, a small soluble heme protein with a molecular mass of ca. 12 kDa, is present in all

organisms [89]. Its molecular structure is highly conserved and varies in different eukaryotes by only

few amino acids, indicating a preservation of its function throughout the evolutionary process [120].

In aerobic organisms, cytochrome cis inter alia attached to the inner mitochondrial membrane, where

it acts as an electron shuttle in the respiratory chain by transferring an electron from cytochrome c

reductase (complex III) to cytochrome coxidase (complex IV). Therefore, it can change reversibly

between the ferric and ferrous oxidation state by donating and accepting an electron, respectively

(see section Cytochromes). Due to its important role in bioenergetics and its high stability in solu-

tion, cytochrome chas been intensively studied by various spectroscopic techniques [121–125]. Aim

of many studies was the investigation of fundamental biological electron transfer processes and the

parameters that control these reactions, using cytochrome cas a model protein [127]. To probe the

properties of biological ET under controlled conditions, the protein is attached to a metal electrode

which is able to trigger the redox transitions by applying potentials. To mimic the natural environment

and to prevent protein denaturation, the electrodes are coated with self assembled monolayers. The

chemical composition of the SAMs can vary depending of the goal of study (see section Self Assem-

bled Monolayers). In this respect, a widely used system is an electrode covered with ω-carboxylated

alkanethiols which possesses a negatively charged surface. The interaction of the protein and the

coated electrode is of electrostatic nature. As a result of a symmetric charge distribution, cytochrome

cexhibits a relatively high molecular dipole moment of several hundreds of Debye [128, 129]. Around

the solvent expose heme edge, seven lysine residues are located which are positively charged at phys-

iological pH and thus form a positively charged patch. The adsorption of cytochrome cto the coated

electrode is also assumed to resemble native biological reaction conditions as most of the redox pro-

cesses take place at charged interfaces. For cytochrome c, this is particularly true for example upon

complex formation with cytochrome coxidase [130, 131].

The first studies aiming at the heterogeneous electron transfer kinetics of cytochrome cwere car-

ried out using electrochemical methods [132, 133]. Electron transfer rate constants were determined

according to a method proposed by Laviron [134]. Due to the relatively high distance between

cytochrome cand the electrode, a non-adiabatic slow electron transfer process was expected. Accord-

ingly, by employing different SAMs with decreasing chain length, the heterogeneous electron transfer

rate should increase as a function of the distance, as is expected for long-range electron transfer due

to the increase of electronic coupling strength according to

kET ∝exp(−βr),(4.1)

4. Results

Figure 4.1: The ET rate constants of cytochrome cand azurin as a function of the thickness of the SAM

layer (distance from the electrode). Circles, cytochrome con Ag. Squares, cytochrome c

on Au. Diamonds, cytochrome con Ag coated with a pyridine terminated SAM. Triangles,

azurin on Au coated with a hydrophobic SAM. Reprinted with kind permission from [38].

where βis the tunneling parameter and rthe effective distance to the electrode. A value of 1.1

per SAM methylene (carboxyl-terminated alkanethiols) unit was determined for β. Interestingly, this

value was found to be essentially the same for different immobilized redox proteins [135–140].

In general, the distance dependence of the heterogeneous electron transfer kinetics of cytochrome c

can be divided in two regimes. For far distances, i.e. for SAMs with 10 or more methylene groups,

it is commonly accepted that the kinetics are dominated by electron tunneling. Accordingly, the ET

rates increase from 0.07 s−1to roughly 50 s1when switching from a C15 to a C10 monolayer (alkyl

chain consisting of 15 and 10 methylene groups, respectively) [141, 142]. A reorganization energy of

0.22 eV was determined by overpotential dependent SERR measurements performed on cytochrome

cadsorbed on C15 SAMs and subsequently fitting of equation 2.45 to the data [38]. However, when

further approaching the electrode, no continuous increase of the ET kinetics can be observed. In fact,

the values of the rate constants determined using a C1, C2 and C5 monolayer level off at around 130

s−1,i.e. the ET kinetics become distance independent for SAMs with six or less methylene groups as

shown in figure 4.1. This region is the so-called plateau region. Due to the unexpected behavior, this

region was object in numerous studies aiming to clarify the origin of the plateau. The fact that this

behavior was also observed for a number of other heme and non-heme proteins suggested, moreover,

that the plateau region might be of general relevance in biological ET processes [143–145, 147, 155].

Although a large body of experimental data was collected, there existed for a long time no con-

sensus about the origin of the distance independence of the ET rates. It was commonly accepted

that a change in mechanism is existent and thus electron tunneling is no longer rate limiting for

short protein-electrode separations [148–150]. In this respect, two different interpretations were

postulated. Avila and coworkers proposed a gated electron transfer mechanism [132]. According

to this, the protein binds to the SAM surface in an orientation which is indeed thermodynamically

stable but does not facilitate the electron tunneling due to a weak electronic coupling. To undergo

the redox reaction, the protein has to re-orientate to a position that favors the electron transfer. This

4.1 Electron Transfer Properties of Cytochrome c at Electrochemical Interfaces

re-orientation is assigned to a rate constant that is the same for all distances to the electrode. While

the electron tunneling rate increases significantly with smaller distances, the re-orientation rate is

constant and therefore becomes rate limiting for a sufficiently small protein-electrode interspace.

Furthermore, this interpretation explains why the rate constants decrease with increasing buffer

viscosity and pH [151, 152]. Both factors have a direct negative influence on the re-orientation rate.

High pHs are equivalent to high surface charge densities due to an increase of the deprotonation

degree of the COOH SAM headgroups. The resulting increased electric field strength slows down

the re-orientation rate, according to kre ∝exp(−∆µ·Ef), where ∆µis the difference in the effective

dipole moment of the protein at the initial and the final orientation and Efis the electric field

strength, respectively. The other model describes the plateau region as a result of a transition from

a non-adiabatic to an adiabatic electron transfer process [153]. Although the electron tunneling

probability is very high, the relaxation of the surrounding medium is comparably slow and thus dom-

inates the observable ET kinetics. This transition from non-adiabatic to adiabatic ET takes place at

small electrode distances where the electronic coupling strength is sufficiently high [153, 154, 156].

Conducted experiments, where the heme group is directly wired to the electrode by coordination of

a pyrdine group to the iron central atom, revealed evidences for the validity of this model [157].

However, all the experimental data supported one or the other model only in an indirect man-

ner. Finally, using Q band excited SERR spectroscopy, it was possible to give the first tentative

answers to the longstanding question about the origin of the plateau region, by probing the protein

re-orientation directly [158]. The approach is based on an exploitation of SE Raman selection rules.

The re-orientation becomes noticeable through changes in the relative intensity of bands of different

symmetry. The reason for this intensity change is the different relative orientation of the heme group

with respect to the surface normal, i.e. the direction of the enhanced electric field vector. Vibrational

modes of the heme group with different symmetry gain surface enhancement due to different entries

of the polarizability tensor α.

αpor =



αxx αxy αxz

αyx αyy αyz

αzx αzy αzz 

(4.2)

In an adsorption geometry as indicated in figure 4.2 in which the green plane represents the heme

group attached on a (much bigger) roughened Ag surface, all entries of the tensor with xcharacter

gain surface enhancement since they are positioned in parallel direction to the enhanced electric field

vector [32, 159] (T direction). In a D4happroximation of the heme group, vibrational modes with

A1gand B1gsymmetry gain enhancement due to the matrix elements αxx,+αyy,αzz and αxx,−αyy,

respectively [74]. If we now assume a starting geometry as shown in figure 4.2 and expect a slow

re-orientation to a configuration where the heme plane is oriented in a parallel way to the surface,

i.e. x,yplane perpendicular to T, then, as a result, the intensity of the B1gmodes will consequently

decrease stronger than the intensity of A1gmodes.

The reason is the extra αzz entry of the latter symmetry. For increasingly parallel orientations, the

zaxis (in heme coordinates) becomes more and more Tlike and thus will gain increasing enhance-

ment. As a result, the intensity ratio of B1gto A1gmodes will be higher if the heme plane is oriented

perpendicular to the surface and will become smaller if the angle between heme plane and surface

normal (in Tdirection) increases. These considerations hold strictly for non-resonant conditions.

This symmetry-based approach was successfully applied using Q band spectra of cytochrome c,

where the rigorous resonance Raman selection rules are weakened [158]. As a result, it is concluded

that orientational dynamics control the overall electron transfer properties. The local electric field

strength determines the mean orientation and the mobility of the protein at the interface. The mea-

sured ET kinetics are concluded to be a convolution of both, the dynamic re-reorientational process

4. Results

Figure 4.2: Model for determining the relative orientation of the heme with respect to the electrode

surface normal (T). x,yand zdenote the heme (green plane) coordinates while T, R, and S

represent the electrode coordinates. The enhanced electric field vector is parallel to T.

and the tunneling probability inherent in each protein orientation.

Although the direct detection of the orientational dynamics constitutes a big step towards under-

standing the biological interfacial ET, substantial questions still remain. In the first project, the prop-

erties of protein surface dynamics were investigated. For that issue, cytochrome cwas attached cova-

lently to a SAM surface by forming amide bonds between the COOH headgroups of the SAM and

the NH2groups of the lysine residues. The effect of a covalent immobilization on the electron trans-

fer kinetics was studied using the approach proposed by Kranich et al, and the measurements were

supported by calculations.

In a second work, kinetic studies of cytochrome cimmobilized on short SAMs were performed and

the results were combined with results obtained from SEIRA measurements, which probes the relax-

ation of the protein backbone. As a crucial parameter, the interfacial electric field could be identified.

It was found that electric field effects modulate strongly the overall ET kinetics by influencing pro-

tein motion, electron tunneling probability and also hydrogen bond re-arrangements.

The obtained results afford a qualitatively consistent description of the interfacial ET process of

cytochrome c.

4.2 Tyrosine Nitrated Cytochrome c - Role in Apoptosis

4.2 Tyrosine Nitrated Cytochrome c- Role in Apoptosis

In contrast to necrosis, the traumatic cell death due to external forces such as injury, apoptosis denotes

the programmed cell death self-initiated by the cell system. The wanted dieback of body cells may

be irritating at the first glance but is of high importance for many biological processes and a crucial

component of life. Apoptosis is mainly employed by multicellular organisms to remove malfunc-

tioning or dispensable cells and plays a role during the embryonic growth, e.g. for differentiation of

extremities and brain development [160, 161]. As a result of apoptosis, the affected cells suffered

shrinkage, plasma membrane blebbing, chromatin condensation and nuclear membrane breakdown.

These cells are subsequently detected and engulfed by phagocytes to avoid an affection to neighbor-

ing healthy cells by the released chemical compounds. A disregulation of the apoptotic function will

finally lead to disease or death of the organism. Cancer, the uncontrolled growth of body cells, is

often mentioned in this respect.

On a molecular level, there are different forms of apoptosis. The most well-known form is the

caspase-dependent (caspase are Cys proteases) apoptosis that may occur via two different pathways:

the extrinsic and the intrinsic pathway [162]. In the extrinsic pathway, the apoptotic stimuli are rec-

ognized extracellularly by a transmembrane protein which acts as death receptor and finally induces

the death-inducing-signalling complex (DISC). The intrinsic pathway, also known as mitochondrial

pathway, is activated by apoptotic inducing factors within the cell leading to permeability of the

outer mitochondrial membrane and a subsequent release of mitochondrial proteins to the cytosol.

Cytochrome cis one of these proteins [163, 164]. Under normal circumstances, cytochrome cis

attached to the inner mitochondrial membrane in the double membrane interspace, where it acts

as an electron carrier to shuttle electrons from complex III to complex IV in the respiratory chain

(compare section Cytochromes). The first experiments pointing at the double role of cytochrome c

in life and death of cells were conducted around 1996 [165, 166]. Further studies revealed that once

cytochrome cis released to the cytosol, it binds to the oligomer Apaf-1, forming a complex that,

in turn, activates the procaspase-9 (apoptosome) to start the apoptotic caspase-dependent reaction

cascade [164, 166]. A more detailed description is given in the references [160, 161].

Before cytochrome ccan be released, a prior detachment from the inner mitochondrial membrane is

necessary. Under normal conditions, it was found that a high fraction of the protein is strongly bound

to the anionic phospholipid cardiolipin via electrostatic interactions [167, 168]. The mechanism of

detaching of cytochrome cfrom cardiolipin is still under debate.

One possibility is the oxidation of cardiolipin to reduce its negative charge and therefore the

binding affinity to cytochrome c[169–171]. This oxidation process can be accomplished either by

reactive oxygen species (ROS) present due to the ongoing respiratory process or by the cytochrome

c-cardiolipin complex itself. The binding promotes a change in the conformation of the protein

allowing oxygen to access to the otherwise closed heme site [172]. Also possible is an effect of the

electric field present at the membrane interface and promoted by the high negative charge of the

cardiolipin that induces a configuration change of the oxidized cytochrome cfrom a native 6cLS

Met/His ligation to a 5cHS /His ligation, where the sixth coordination place remains vacant for

oxygen binding and subsequent peroxidation reactions [173–175]. Other cytochrome cmodifications

are adducts formed upon reaction with reactive nitric oxides (RNS). Cytochrome chas been shown

to be nitrosylated at the heme iron atom during the apoptosis [176]. Beside this, nitrated tyrosines

are also possible reaction products. Tyrosine nitration is proposed to be one of the initial steps of the

transformation of cytochrome c’s function from an electron carrier to a signal transducer [177–180].

Human cytochrome cexhibits five different tyrosines, the heme near buried tyrosine Y48 and Y67

and the solvent exposed Y46, Y74 and Y97.

In the study conducted in this thesis, the effects of different tyrosine nitrations on the heme site

of cytochrome cwere investigated. Single tyrosine mutants for each five tyrosines were prepared by

4. Results

specific Tyr to Phe substitution of four tyrosine groups. The remaining tyrosine residue was subse-

quently nitrated. The thus treated mutants were first investigated by means of resonance Raman to

estimate the effects of tyrosine nitration on the heme structure. To probe the effects of electric fields

on the stability and the conformational equilibria, the nitrated mutants were immobilized on a SAM

coated electrode and studied by means of surface enhanced resonance Raman spectroscopy. The

results indicate that the heme pocket destabilizations caused by nitration and by electric fields occur

via different driving forces and mechanisms. Moreover, it is concluded that the higher accessibility

of the heme site, caused by nitration, facilitates the peroxidase activity of the protein.

4.3 Interfacial ET of Outer Membrane Cytochromes Embedded in Biofilms of Geobacter

4.3 Interfacial ET of Outer Membrane Cytochromes Embedded in

Biofilms of Geobacter

The first metal reducing bacteria strain was discovered in 1987 where it was isolated from sediments

of the Potomoc river, USA [181]. These bacteria, named therefore with the suffix metallireducens,

were found to be capable of oxidizing small organic compounds to carbon dioxide using iron oxides

as electron acceptors [182]. The application potential of these bacteria was soon realized. In the fol-

lowing years, many other strains were discovered exhibiting similar abilities [183].

Among all of them, two species have been studied most extensively. These are the strains Geobacter

sulfurreducens and Geobacter metallireducens. Beside academic research interests, a lot of efforts

have also been made to employ these bacteria in technical applications [184, 185]. Two major

aspects of the bacteria’s properties are in this respect important. One concerns the potential sub-

strates. Geobacter species have been shown to be able to metabolize (oxidize) many different kinds

of organic compounds [186, 187]. Accordingly, they can be used in environmental restoration pro-

cesses, for example for decomposing harmful organic pollutants such as petroleum or aromatic com-

pounds, both exposing a high threat to the qualtity of groundwater [188, 189]. Also metal ions can be

used as electron donors. This includes especially Fe(II) and U(IV) [191–193]. Geobacter can oxidize

these metals to the higher oxiditation states, i.e. Fe(III), U(VI) and U(V), which is accompanied by

a significantly lowering of the solubility of the compounds. In the case of U(IV), this is of special

interest in terms of binding this highly toxic element for subsequent removal from water or in general

environmental systems [191]. The other aspect concerns the potential electron acceptor. It has been

found that these bacteria can not only grow on iron oxides but also accept a variety of other oxides

and elements as client (i.e. extracelluar electron acceptors). To mention the most important, these are

bulk carbons, tin oxide, silver and gold.

Given a stable food and an insoluble electron acceptor source, Geobacter can even form biofilms of

up to 50 µm thickness above these materials, exhibiting a relatively high substrate consumption rate,

see figure 4.3 [194]. Therefore, a major interest has been focused on the use of Geobacter for pro-

ducing electricity by growing the bacteria on solid electrodes and using their metabolism as electron

generating source [184, 196]. This approach finally led to establishing the field of microbial fuel cells

(MFC)(in bioelectrochemical systems, BES) in analogy to ordinary H2/O2fuel cells [184]. In these

MFCs, the bacteria are grown on a solid anode material to yield stable biofilms and subsequently

exploiting their substrate metabolism for electricity generation. On the prime time of MFC develop-

ment, even the American aerospace organisation NASA considered to install MFCs in shuttles to gain

electricity out of the inevitably accumulating waste water during space missions. There have also

been efforts to upscale these MFCs to be used industrially [197]. However, a kind of disenchantment

seems to be slowly manifestating in the MFC community lately. It seems that the performance in

terms of electricity generation of MFCs are far too low to be used for "real world" applications [197].

To reach a higher current output, two approaches are in principle possible. On the one hand the anode

material can be optimized to enhance the bacteria/electrode interactions and thus allowing possibly

higher current density outputs. On the other hand genetic manipulations can be applied to increase

the overall bacterial turnover activity [194].

However, both approaches require a deep understanding of the processes ongoing in the bacteria. In

this respect, particularly the electron transfer mechanism through the bactaria colony, i.e. the biofilm,

and finally to the electrode (insoluble acceptor) is an important factor. Accordingly, there have been

many studies carried out addressing especially this issue. A lot of interesting information has been

gathered but still an overall clear picture is missing.

4. Results

Figure 4.3: SEM images of G. sulfurreducens growing on a gold electrode. Magnification: 75-20 000

x. A. Biofilm attached to the surface, partially peeling off. B. Closeup of Figure 3A where

the biofilm was attached to the electrode surface. Cand D. Closeups of Aat the edge of the

Society.

Electron Transfer Properties

Most of the studies on the electron transfer properties referred to the strain Geobacter sulfurreducens.

Although there are a lot of unknown parameters, it could be concluded that an efficient electron

transfer of Geobacter requires a number of certain proteins. For extracellular electron transfer, it

seems that Geobacter possesses a variety of redox active outer membrane cytochromes (Omcs)

which are located either in or on the circumferum of the cell’s outer membrane [198]. These Omcs

are multi c-type heme proteins whereby the exact number of heme groups within a protein can

differ from type to type. The most important Omcs discovered so far are the OmcB, OmcS and

OmcZ [194]. Each protein is associated with another function. For example, OmcB contains twelve

heme groups and is mainly embedded in the outer membrane with a small part exposed [199]. It

is assumed that this protein plays a role in the direct electron transfer between the bacterium and

an acceptor. In contrast, OmcS can be found on the outer membrane of bacteria cells [202]. The

exact role of this six heme groups containing protein is not known yet. OmcZ is also located on

the outer membrane and will be accumulated heavily at the bacterium/solid interface if the electron

acceptor constitutes an insoluble solid anode. In that case, Geobacter tend to form thick biofilms. It

was found that OmcZ is crucial for the electron transfer through the biofilm (high current densities

for biofilm/anode systems) but not for reducing small soluble electron acceptors [203]. The general

determination of respective roles of certain Omcs is very difficult as one has to rely of gene deletions

methods. The bacteria, however, can replace easily the removed Omc type by expressing another one

instead, complicating the assignment severely. Overall, the accumulated knowledge about the Omcs

is still very limited [194].

In case of Geobacter biofilms, it is important to distinguish between major electron transport steps.

One step is the heterogeneous electron transfer reaction at the interface of bacterial film and solid

4.3 Interfacial ET of Outer Membrane Cytochromes Embedded in Biofilms of Geobacter

Figure 4.4: Transmission electron microscopy picture of G. Sulfurreducens cells. The filament

strucutres constitute the pili protein network produced by the bacterium. Scale bar, 0.2 µm.

Reprinted with permission from Macmillan Publishers Ltd: Nature. Figure from [201].

support. It is commonly accepted that Geobacter can transfer directly electrons to the acceptor, in

contrast to other species which employ a variety of electron shuttles and mediators for this purpose.

For that, it is likely to assume that a very close (tunneling) contact between bacterium and acceptor

is needed in order to enable heterogeneous ET. In previous studies, a number of Omcs are proposed

to play a role in this process, e.g. the aforementioned OmcZ [203].

The other electron transfer step concerns the shuttling of electrons through the biofilm, which

includes also the transfer of electrons from out of the inner cell of distant bacteria, where chemi-

cal conversion takes place, and subsequently transporting through the biofilm matrix and finally to

the electrode. Since a biofilm can have a thickness up to 50 µm, there are bacteria cells that are not

in contact with the electrode surface. However, it was observed that the current production increases

with increasing biomass present in the biofilm, suggesting that a) cells more remote from the elec-

trode are in electronic contact and can transfer electrons and b) hence the biofilm has to exhibit a

certain electron conductivity [204].

In this respect, two major approaches are proposed to explain the intrabiofilm electron flow. The

more traditional approach involves the electron transport through (matrix-)cytochromes, abundantly

present in the biofilm as it shines red as a result of the electronic absorption properties of the

heme groups. The electron transport mechanism was proposed to be similar to that in hydro-gels or

other biological systems (e.g. cytochrome coxidase, PSI&II) involving electron hopping mechanism

between distant redox centers, i.e. cytochromes [205, 206]. In contrast to this, the other approach

states that pili constructs, inherent in biofilms, play a major role in the electron transport cascade

[204]. The pili structures constitute a major discovery and are found to be produced in particular

by Geobacter Sulfurreducens when grown in biofilms. In SEM pictures of Geobacter biofilms, see

figure 4.4, these filament-like pili constructs, mainly consisting of the structure protein pilA, can be

easily identified as they are abundantly produced and form network-like structures to connect distant

bacterial cells with each other [207]. Although previous studies suggested that these pili are mainly

insulators, recent studies carried out indicate a high conductance along the pili. In fact, the conductiv-

ity was found to increase exponentially with decreasing temperature similar to metal systems [208].

Therefore, it was concluded that these pili may play also a role in the electron transport chain through

the biofilm matrix. Moreover, associated with the pili are the OmcS and OmcZ which both seem to

4. Results

be necessary in order to obtain high current densities in anode/biofilm systems [202]. Consequently,

it was propsed by Lovley et al. that these pili act as electron channels. The electrons are transported

possibly via π-πstack interactions, as no cytochromes are present along the pili.

However, to prove or disprove the validity of one or the other model, way more experimental studies

have to be carried out. The current art of knowledge on that topic is far from being complete.

Spectro-electrochemistry of electro-active Biofilms of Geobacter Sulfurreducens

The simplest method for characterizing the electro-activity of biofilms is cyclic voltammetry (CV).

Here, the electrode potential is linearly swept and the resulting currents measured and plotted as a

function of cell potential. The CV experiment can be performed for biofilms (grown on the working

electrode) in two modes, in the presence or absence of substrate. Each condition delivers different

information about the system. In the presence of substrate, the voltammogram shows the typical

turnover signal, a quasi-sigmoidal current trace [209, 210]. It could be shown that the turnover

activity, i.e. the total produced current density, depends on the electrode potential and is higher the

more positive the applied electrode potential is [211]. Under non-turnover conditions, i.e. in the

absence of substrate, the voltammogram obtained for very low scan rates (1 mV/s) shows a variety of

unique peaks. These peaks result from electrode nearby redox centers whose reactions were triggered

by the changing electrode potential. It is likely to assume that the nature of these redox centers might

include Omcs that are expressed by the bacteria in order to allow electron exchange with the bulk

metal (vide supra). Unfortunately, CV cannot provide information about the chemical constitution of

the reaction partners involved. To investigate further the compounds involved in the electron trans-

port cascade, CV and in general electrochemical techniques need to be coupled with spectroscopic

methods. The yielded combination, spectro-electrochemistry, has proven to be a powerful method,

particularly for investigating surface-confined redox active biological systems [38, 40, 212, 213].

For biofilms that contain a lot of heme proteins such as plasmic cytochromes and Omcs, SERR spec-

troscopy turned out to be the ideal spectroscopic tool [210]. In a first study by Millo et al., SERR

spectroscopy was successfully applied for investigations of surface-confined Omcs in biofilms of

Geobacter Sulfurreducens. The biofilm was therefore grown on a roughened Ag electrode that

constitutes the SER active support. Interestingly, the bacteria could grow also in the presence of

silver cations, which normally have a disinfectant effect. In fact, the bacterial turnover activity

resulted in high current densities up to 600 µA/cm2. SERR spectra collected from that system,

using a 413 nm excitation which is in resonance with both the heme group and the support, clearly

showed a spectral signature of a c-type heme with two His residues functioning as axial ligands

(bis-His 6cLS). Upon changing the electrode potential, the relative amount of oxidized and reduced

hemes changed indicating that the heme groups are involved in the electron transport reaction /

surface redox reaction. As a result of the electrochemical titration experiments, it could be shown

that, consistent with CV experimental predictions, two kinds of heme containing redox species are

involved in the heterogeneous electron transfer process. These heme groups belong most likely to

two different Omcs of bacterial cells in close contact with the electrode surface. Along with findings

obtained with other spectroscopic techniques such as UV/Vis or SEIRA, it could be conclusively

shown that, as predicted by microbiological methods, cytochromes are involved in the interfacial

electron transport of biofilms [214–218].

As a part of this thesis, the well-established time resolved SERR spectroscopic approach was

applied to investigate the heterogeneous electron transfer properties of the Omcs in close vicinity to

the electrode surface [38]. Similar to the previous study of Millo et al, bacterial biofilms of Geobacter

Sulfurreducens were grown on roughened silver electrodes. Then, potential jumps were applied to

trigger the redox reaction at the working electrode and SERR spectra were recorded as a function of

different delay times following the potential jumps. The interfacial redox process could be resolved

4.3 Interfacial ET of Outer Membrane Cytochromes Embedded in Biofilms of Geobacter

as a function of time and the applied (over-)potential. As a result, we were able to determine different

rate constants for the surface relaxation process. The experiments were furthermore combined with

electrochemical measurements, i.e. CV and chrono-amperometry, and kinetic simulations to support

the Raman results. The overall obtained picture indicates that the heterogeneous electron transfer

seems to be the rate limiting step of the biofilm/electrode ET process, while the intra-biofilm electron

shuttling (at least at a distance close to the electrode) is much faster. Moreover, we obtained evidences

that Omcs may act as electron gates and therefore functioning as electronic link between the biofilm(-

matrix) and the solid anode.

4.4 Surface Enhanced Raman Active materials - Pt Coated Ag Electrodes

4.4 Surface Enhanced Raman Active materials - Pt Coated Ag

Electrodes

SER spectroscopy has been constantly further developed over the last decades. Although nowadays

this technique is already employed in various kinds of scientific studies, the limits of its potential

are still not reached. The largest progress in this field has been made since the middle of the 1990s

with the beginning of controlled design of SE active support materials. These supports are crucial

for the sensitivity of the method, since their electronic properties determine the surface enhancement

and therefore the signal intensity increase. The efforts made in searching for different nanostructures

and geometries producing the highest field enhancements had finally led to an own field of research

named plasmonics [219].

Most of the studies focus on the coinage metals silver, gold and copper. All these three belong to the

free-electron (Drude) metals, which can be excited resonantly using visible light to generate intense

surface plasmon resonances [220]. Accordingly, their field enhancements are very high, so that they

constitute suitable materials for SER studies. Other metals such as the transitions elements Pt, Ti and

Ru, however, show only weak surface enhancements with plasmon resonance frequencies expected

to lie in the UV region [221, 222]. Their Fermi energy levels are in the region of the dbands that

are subject to strong mixing with sorbitals, and thus interband transitions become very likely. As a

result, surface enhancement is almost depleted completely [223–225].

This constitutes a major drawback and is a clear restriction of SER spectroscopy. Specifically, the

metals like Rh, Ru and Pt are more widely used in technical applications such as heterogeneous

catalysis, semiconductors and energy conversion systems. Elucidation of the underlying processes

would benefit greatly from an application of SER spectroscopy to non-coinage metals.

There have been efforts made to expand the applicability of SER spectroscopy in this respect. Among

those, three different approaches can be distinguished. In the first approach, the surface roughness of

these metals is increased by a factor of up to 200 via electrochemical roughening procedures as also

applied in this work for silver electrodes [220]. The massively increased surface area allows binding

of a significantly higher number of molecules and thus enables probing them under off-resonance

conditions [226–230]. Admittedly, this approach represents no kind of SER spectroscopy since no

surface enhancement is involved, but corresponds to surface Raman spectroscopy. Another approach

exploits the weak surface enhancement effect of these metals, manifesting upon irradiation with soft

to hard UV light [231, 232]. Tian et al. managed to obtain moderate signals by performing UV-SER

spectroscopy in solution [220]. Possible drawback of this method is the high energetic light source,

which may introduce side reactions and decomposition of probe molecules. In the third approach, a

non-SER active material is deposited on a SER active support. The plasmons excited in the lower

layer can induce SER activity in the outer layer, given that certain requirements are fulfilled. With

this method, considerable SE can be gained [233].

There are different ways of generating such a bilayer composite of two materials with different elec-

tronic properties. Usually, the lower support is a highly SER active material such as silver or gold.

The top layer can be deposited through various techniques. Vapor deposition and electrochemical

deposition methods can be used, if the second layer should be another metal like Pt, Pd, Rh, Fe

etc. In case the second layer is a composite like TiO2or SiO2, standard nucleation techniques are

applicable. The obtained signal intensities are different depending on the applied technique. For

example, bimetallic devices generated via direct electrochemical deposition exhibit only moderate

signal intensities of probes attached at the outer layer, while rough Ag coated with a thin TiO2by

evaporation induced self-assembly (EISA) yielded comparably high signals also for thicker oxide

layers.

Recently, Feng et al reported a method to fabricate bimetallic Au-Ag hybrid devices that exhibit only

low intensity loss measured at the outer metal [233]. Here, a solid silver electrode is first roughened

4. Results

Figure 4.5: Left. SEM picture of a Pt island film on a rough Ag electrode. Center. Schematic represen-

tation of the hybrid device. The rough Ag electrode is depicted in grey. The yellow layer

represents the dielectric spacer. The dark grey layer on top represents the deposited Pt film

(here closed film). The probe molecules are mercaptopyridine and the protein cytochrome

c.Right. Modeled Pt-Ag hybrid device (Pt island film with defects) in field calculations.

The color code on the right displays the intensity of the near-field. Reprinted with kind

to yield a SER active surface. Before depositing the top metal via metallation by electrochemical

reduction from metal salts in solution, a dielectric spacer is coated on the lower layer. With this pro-

cedure, relatively high signal intensities of probe molecules on the outer metal layer can be obtained.

Additionally, it could be shown that the outer metal kept its original electrochemical and chemical

properties, which is crucial since the outer metal represents the platform for the surface-confined

processes to be studied.

Inspired by these results, a similar method was employed to generate Pt-Ag hybrid devices (Figure

4.5). Pt was chosen due to its high relevance, especially in heterogeneous catalysis like CO conver-

sion and hydrogen formation. The overall goal was to fabricate a solid support to probe adsorbed

molecules on the Pt surface at different stages of a catalytic reaction. The device should provide high

SER signal intensities and should be easy to handle and stable even at relatively high temperatures

up to 500 ◦C.

In the final part of this thesis, the first results on the Pt-Ag hybrid device are reported. Here, the

main focus lied on the overall signal performance, i.e. the relative enhancement factors obtained by

using spacers of different kind and length. The experiments were supported by calculation. As a

crucial point, it could be shown that films with defects facilitate high SER signal intensities while

closed films tend to deplete SER activity. Intense SE(R)R signals of probe molecules, attached at the

outer Pt layer, were obtained. The signals were only slightly weaker compared to signals of molecules

directly adsorbed on the Ag. Although not included in the publication, first tentative experiments

indicated that also good quality signals of CO and H2, adsorbed on Pt, can be obtained.

5 Conclusions and Outlook

In this thesis, RR and SERR spectroscopy was successfully applied to investigate heme contain-

ing proteins. Aim of the studies was to obtain insights into the function and mechanism of these

biomolecules. In particular, the heterogeneous electron transfer properties of horse heart cytochrome

cand the electron transfer of outer membrane cytochromes embedded in biofilms of Geobacter

Sulfurreducens were investigated using SERR and TR-SERR spectroscopy. Furthermore, RR spec-

troscopy was used to study the effects of nitration of tyrosines in human heart cytochrome con the

integrity of the heme site structure in order to estimate the effects of this posttranslational amino

acid modification on the properties of the protein with respect to its role in the apoptosis. The results

obtained in the individual subprojects provide novel insights into the biophysics of these proteins. In

summary, it could be demonstrated that the different types of Raman spectroscopy constitute valu-

able techniques for investigating biological systems. The last part of the thesis was dedicated to the

development of the SER methodology, aiming at expanding the technique to study processes on metal

surfaces other than Ag or Au.

Electron Transfer Properties of Cytochrome cat Electrochemical

Interfaces

The studies allowed disentangling the complex redox process of cytochrome cat electrochemical

interfaces. It was shown that the heterogeneous ET of cytochrome cis a convolution of at least four

different fundamental steps. These steps are electron tunneling coupled with the protein reorganiza-

tion, protein surface dynamics and rearrangement of hydrogen bond networks at the interface and

possibly also inside the protein. Moreover, it was found that the interfacial electric field is a crucial

parameter that controls the overall ET process by affecting each single step. On the basis of that, it

was possible to provide a qualitatively consistent picture of the interfacial ET process that explains

present and previous findings. It is also concluded that electric fields may be of general relevance for

interfacial processes in biology, beyond the ET of cytochrome c.

Although the findings represent a significant gain of knowledge about the interfacial ET, a detailed

picture of this issue is far from being complete. In this respect, also a quantitative description of the

ET process would be needed, which constitutes a challenging task and would require new experimen-

tal approaches and strategies. Finally, it would be interesting to move on from model systems, i.e. the

adsorption on a SAM coated electrode, to natural reaction complexes and to study for example the

ET transfer within the CcO/cytc complex, using the knowledge accumulated so far as a basis for the

interpretation.

Tyrosine Nitrated Cytochrome c

It was shown that the Tyr to Phe substitution had less impact on the heme pocket but led to significant

shifts of the redox potential of the Fe2+/3+transition, depending on the substituted tyrosine residue.

These shifts were additive, indicating that there might be long-range electrostatic perturbation effects

on the redox potential. The nitration of the remaining Tyr caused severe changes in the ligation pat-

tern and led to formation of heme states in which the Fe-S(Met80) bond is broken. It was found that

5. Conclusions and Outlook

a 5cHS and also a 6cLS species, where the Met80 is replaced by a Lys, are formed. Furthermore, no

correlation between increased peroxidase activity and relative HS content could be identified, indi-

cating that the increased H2O2accessibility may be more important for the peroxidase activity than

the vacant coordination sphere. Upon electrostatic immobilization on a SAM coated electrode, signif-

icant destabilizations of the heme pocket were noted for both the mono-Tyr and the nitrated proteins.

In contrast to the situation in solution, a His-His 6cLS heme species was found. These findings indi-

cate that the destabilizing effects caused by the interfacial electric field and by nitration of Tyr have

different structural consequences, supporting the view that either nitration or binding to cardiolipin

and possibly also both in concert are responsible for the alteration of the function of cytochrome c.

With regard to further possible Raman investigations of apoptotic cytochromes, cytochromes isolated

from the cytosol of dying cells during different stages of apoptosis, should be investigated. These

experiments would clarify which type of ligation the cytochromes exhibit after leaving the mitochon-

drium and if the alteration of the heme site, i.e. ligation pattern and electrostatics, is required for

the apoptotic function of cytochrome cin the cytosol. Further possible investigations may also focus

on other posttranslational tyrosine or protein modifications such as phosphated tyrosines or oxidized

methonines.

Interfacial ET of Outer Membrane Cytochromes Embedded in Biofilms

of Geobacter

The study reveals a first picture about the heterogeneous electron transfer of the surface confined

heme proteins. Here, the Omcs located at the biofilm/electrode interface act as electron ’gates’ and

provide the electric connection between the biofilm and the electrode. The heterogeneous ET of

these surface confined Omcs is relatively slow (0.03 s−1) compared to the intramolecular ET with

the first layer of redox partners located in the bulk biofilm (ca. 40 times faster), and therefore can be

considered as the bottleneck process of the overall ET cascade. Furthermore, pure electron tunneling

is most likely not the mechanism of electron transfer at the interface.

This study demonstrates the applicability of SERR and TR-SERR spectroscopy for investigating

biofilms and other MFC systems. This constitutes a major progress since usual investigations of the

biofilm activity and kinetics rely mainly on electrochemical methods, which are inherently insensitive

to the nature of the probes. Future experiments using SERR spectroscopy may address particularly

the interface reactions, aiming to identify the exact mechanism of the heterogeneous ET process, e.g.

hopping or coupled ET. Also the SERR/TR-SERR spectroscopic approach could be applied to other

bacteria strains, for example the metal reducing bacteria strains of Shewanella.

Surface Enhanced Raman Active materials - Pt Coated Ag Electrodes

The fabricated Pt-Ag hybrid device shows good SE(R)R performance for the probe molecules mer-

captopyridine and cytochrome c. The coating with dielectric spacers effectively isolates the Ag sup-

port, blocking the direct adsorption of probe molecules on the Ag. Furthermore, it was found that the

SER signal intensity did not depend on the nature of the outer metal and only weakly on the thickness

of the dielectric layer. The crucial parameters are the enhancement of the Ag support and the kind of

metal film at the outer layer. From electromagnetic field calculations it could be concluded that Pt

island films that exhibit defects and holes, provide a significantly stronger induced surface enhance-

ment at the outer layer and thus higher SER signal intensities. The first tests of the hybrid device for

probing adsorbed CO and H2were promising, and good signals of the CO stretching and of the Pt-H

stretching modes were observed.

Future studies may focus on the performance of the Pt-Ag hybrid device at high temperatures up to

5. Conclusions and Outlook

800 K. Additionally, it would be desirable to replace the electro-deposition of Pt by another method,

e.g. vapor deposition onto a patterned rough Ag surface, which would allow for more controlled

design of the Pt surface structures.

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Thermal Fluctuations Determine the Electron-Transfer

Rates of Cytochrome cin Electrostatic and Covalent

Complexes

Ly, H. K.; Marti, M. A.; Martin, D. F.; Alvarez-Paggi, D.; Meister, W.; Kranich, A.;

Weidinger, I. M.; Hildebrandt, P.; Murgida, D. M. Thermal Fluctuations Determine the

Electron-Transfer Rates of Cytochrome c in Electrostatic and Covalent Complexes.

ChemPhysChem 2010, 11(6), 1225–1235.

DOI: 10.1002/cphc.200900966

Thermal Fluctuations Determine the Electron-Transfer

Rates of Cytochrome c in Electrostatic and Covalent

Complexes

Hoang Khoa Ly,[a] Marcelo A. Marti,[b] Diego F. Martin,[b] Damian Alvarez-Paggi,[b]

Wiebke Meister,[a] Anja Kranich,[a] Inez M. Weidinger,[a] Peter Hildebrandt,*[a] and

Daniel H. Murgida*[b]

1. Introduction

Direct electrochemistry of redox metalloproteins immobilized

on electrodes coated with biocompatible or biomimetic films

is an active field of fundamental and applied research. On the

one hand, electrochemical and spectroelectrochemical meth-

ods can afford a great deal of information on the electron-

transfer (ET) mechanisms and dynamics of the anchored pro-

tein that can contribute to the understanding of its function-

ing in vivo.[1–3] For instance, protein dynamics has been recent-

ly recognized as a key factor in controlling or limiting inter-

and intraprotein ET reactions.[4–15] However, in most cases the

complexity of the systems impairs direct observations of con-

formational gating, configurational fluctuations, or rearrange-

ment of protein complexes under reactive conditions. In this

context, the combination of suitable spectroelectrochemical

techniques with simplified model systems, such as proteins im-

mobilized on biomimetic electrodes, can greatly contribute to

the elucidation of the biophysical fundamentals in better

detail. On the other hand, the knowledge gained from these

studies is essential for the rational design of protein-based

technological devices such as biosensors and biofuel cells.[16–18]

The most widely used electrode coatings are self-assembled

monolayers (SAMs) of single or mixed alkanethiols containing

w-functional groups that are chosen according to the protein’s

surface properties.[2,3] One of the specific advantages of this

approach is to facilitate the determination of ET rate constants

kET as a function of distance by simply varying the chain length

of the thiols, as first shown by Chidsey.[19] In principle, the het-

erogeneous ET reaction for such systems is expected to follow

a nonadiabatic mechanism, and thus its rate can be described

in terms of the high-temperature limit of the semiclassical

Marcus expression, integrated to account for all the electronic

levels eof the metal electrode contributing to the process

[Eq. (1)]:[20]

kET ¼p

hjVj21

ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

plkBT

1

exp lþðeFeÞþehðÞ

4lkBT



1þexp eeF

ðÞ=kBT½

ð1Þ

where 1(e) is the density of electronic states in the electrode,

and eFthe energy of the Fermi level. The applied overpotential,

The heterogeneous electron-transfer (ET) reaction of cytochro-

me c (Cyt-c) electrostatically or covalently immobilized on elec-

trodes coated with self-assembled monolayers (SAMs) of w-

functionalized alkanethiols is analyzed by surface-enhanced

resonance Raman (SERR) spectroscopy and molecular dynamics

(MD) simulations. Electrostatically bound Cyt-c on pure carbox-

yl-terminated and mixed carboxyl/hydroxyl-terminated SAMs

reveals the same distance dependence of the rate constants,

that is, electron tunneling at long distances and a regime con-

trolled by the protein orientational distribution and dynamics

that leads to a nearly distance-independent rate constant at

short distances. Qualitatively, the same behavior is found for

covalently bound Cyt-c, although the apparent ET rates in the

plateau region are lower since protein mobility is restricted

due to formation of amide bonds between the protein and the

SAM. The experimental findings are consistent with the results

of MD simulations indicating that thermal fluctuations of the

protein and interfacial solvent molecules can effectively modu-

late the electron tunneling probability.

[a] H. K. Ly,+W. Meister, Dr. A. Kranich, Dr. I. M. Weidinger, Prof. P. Hildebrandt

Technische Universitt Berlin, Institut fr Chemie

Str. des 17. Juni 135, Sekr. PC14, Berlin (Germany)

Fax: (+49)30-31421122

E-mail: hildebr[email protected]lin.de

[b] Dr. M. A. Marti,+D. F. Martin,+D. Alvarez-Paggi, Prof. D. H. Murgida

Departamento de Qumica Inorgnica, Analtica y

Qumica Fsica INQUIMAE-CONICET

Facultad de Ciencias Exactas y Naturales

Universidad de Buenos Aires, Ciudad Universitaria

Pab. 2, piso 1, C1428EHA Buenos Aires (Argentina)

Fax: (+54)11-4576-3341

E-mail: dhmurgid[email protected]

[+]These authors made equal contributions to this work and are thus listed in

alphabetical order.

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cphc.200900966.

ChemPhysChem 2010, 11, 1225 – 1235  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1225

reorganization energy, and magnitude of the electronic cou-

pling are denoted by h,l, and jVj, respectively. The remaining

parameters have the usual meaning. Equation (1) can be ex-

pressed in a simplified form by approximating the Fermi distri-

bution law as a step function [Eq. (2)]:

kET p

hjVj21erfc lþeh

ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4lkBT

! ð2Þ

where erfc(z) is the complementary error function.

The electronic coupling jVjdecays exponentially with in-

creasing separation of the redox center from the electrode,

and therefore Equations (1) and (2) predict an exponential dis-

tance dependence of kET, that is, decreasing rate with increas-

ing chain length of the alkanethiols. This prediction has been

verified for a variety of redox proteins immobilized on different

types of SAM-coated electrodes, including CuAcenters, azurin,

iso-cytochrome c and cytochromes c,c6,andb562, among

others.[21–26] The average tunneling decay parameter bob-

tained for the different proteins is about 1.1 per methylene

group of the thiols.

A common feature encountered for all cases studied so far is

that the exponential variation of the apparent ET rate constant

kapp

ET is only verified when the proteins are attached to relatively

long chain tethers, usually containing nine or more methylene

groups. In contrast, for thinner SAMs, kapp

ET becomes distance-in-

dependent, thus suggesting a change of the reaction mecha-

nism. This “unusual” distance dependence has been most ex-

tensively studied for mammalian cytochrome c (Cyt-c) by sev-

eral groups employing different approaches. Most of the work

refers to Cyt-c electrostatically adsorbed to electrodes coated

with SAMs including w-carboxyl alkanethiols.[23,27–34] In addition,

Waldeck and co-workers have studied the ET kinetics of Cyt-c

coordinatively bound to electrodes coated with mixed SAMs in

which one of the thiol components contained a tail group able

to bind to the heme iron atom by displacing the native axial

ligand Met80.[23,35–38] Both modes of immobilization yield a

qualitatively similar “unusual” distance dependence of kapp

ET , al-

though values for coordinative binding are higher due to the

artificially improved electron-transfer pathway. Furthermore, in

both cases the ET rates in the plateau region of the kapp

ET versus

distance plots depend on the viscosity of the solution, which

has been discussed controversially. Niki et al. proposed a gated

mechanism based on a two-state model, in which the thermo-

dynamically stable electrostatic complex Cyt-c/SAM represents

an unfavorable configuration for ET such that protein reorien-

tation is required to generate the electrochemically active

state.[23,32] The model assumes that the rate of reorientation is

distance-independent, while electron tunneling rates vary ex-

ponentially. Therefore, at sufficiently short chain lengths reor-

ientation becomes rate-limiting and kapp

ET reaches a plateau. In

contrast, based on studies of coordinatively bound Cyt-c, Wal-

deck et al. proposed a change from the nonadiabatic regime

at long distances to the friction-controlled (adiabatic) regime

at short distances,[38] which in principle could also apply to

electrostatic Cyt-c/SAM complexes. Time-resolved surface-en-

hanced resonance Raman (TRSERR) spectroeletrochemical ex-

periments showed that coordinative Cyt-c/SAM complexes ex-

hibit a pronounced overpotential dependence of kapp

ET in the

plateau region with a reorganization energy of 0.58 eV, and

thus support the friction model.[35] For electrostatic complexes,

instead, kapp

ET is nearly independent of the applied overpotential

in the plateau region, and thereby points to a process other

than ET that becomes rate-limiting at short distances.[3] By

using Q-band excitation TRSERR[27,30] and molecular dynamics

(MD) simulations,[27,39] we have provided the first direct evi-

dence that this gating mechanism is related to protein reorien-

tation in search of efficient electron pathways. The resulting

model, however, differs from that originally proposed by Niki

et al., since reorientation rates have been found to be modu-

lated by the interfacial electric field, which in turn varies with

the chain length of the SAMs. Moreover, we have shown, both

experimentally[2,3,40] and theoretically,[41] that electric fields of

the magnitude estimated for short SAMs as well as for biologi-

cal interfaces, are able to modulate not only the ET dynamics

but also the structure and function of the protein. These find-

ings support the hypothesis that the transmembrane potential

in mitochondria may regulate the ET-driven proton-pumping

activity of cytochrome coxidase (CcO) through electric field

dependent interprotein ET from Cyt-c to CcO. Furthermore, the

interfacial electric field may constitute the switch for the transi-

tion from the redox to the apoptotic function of Cyt-c.

In a recent electrochemical study, Davis et al. reported that

the ET kinetics of Cyt-c cross-linked to mixed SAMs composed

of COOH- and OH-terminated alkanethiols exhibits identical

distance dependence to electrostatic complexes with the same

SAMs, and is qualitatively similar to that of electrostatic com-

plexes with pure w-carboxyl alkanethiol SAMs.[42] Since cova-

lent attachment is expected to restrict protein mobility, these

results cast some doubts on the general validity of the electric

field dependent protein-dynamics model proposed previous-

ly.[2,30]

The present work is dedicated to elucidating this contradic-

tion. Using TRSERR and MD simulations we have performed a

comparative study of the ET properties of Cyt-c in electrostatic

and covalent SAM/Cyt-c complexes with single-component

and mixed SAMs. The results suggest that in all cases the ET

rates are modulated by low-amplitude motions of the protein

and thermal fluctuations of interfacial water molecules, which

in turn are influenced by the interfacial electric field.

2. Results and Discussion

2.1. Structural and Redox Equilibria of the Immobilized

Protein

Nanostructured Ag electrodes were coated with SAMs of differ-

ent compositions for subsequent immobilization of horse heart

Cyt-c. The different films include single-component SAMs of 6-

mercaptohexanoic acid (C5-COOH) and of 11-mercaptoundeca-

noic acid (C10-COOH), as well as mixed SAMs of 1:1 mixtures

of C5-COOH/6-mercaptohexan-1-ol (C5-COOH/C6-OH) and of

C10-COOH/11-mercaptoundecan-1-ol (C10-COOH/C11-OH). All

experiments were carried out at pH 7.0 such that the SAMs ex-

1226 www.chemphyschem.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2010, 11, 1225 – 1235

P. Hildebrandt, D. H. Murgida et al.

hibited a negatively charged surface due to partial dissociation

of the carboxylic acid tail groups, thereby allowing the posi-

tively charged Cyt-c to bind electrostatically, as reflected by

the strong and characteristic SERR signals. The SERR spectra of

ferrous Cyt-c adsorbed on the different SAMs, recorded upon

Soret-band excitation, are identical to the corresponding reso-

nance Raman (RR) spectrum in solution, that is, the structure

of the heme pocket is preserved upon adsorption. Further-

more, the sensitive response of the spectra to the applied elec-

trode potential indicates that Cyt-c undergoes direct electro-

chemistry in all of the SAM/Cyt-c electrostatic complexes inves-

tigated, in agreement with previous findings.[2,3] Within the po-

tential range from 400 mV to about 50 mV (vs. Ag/AgCl), all

SERR spectra can be quantitatively described by a superposi-

tion of the RR spectra of the native state of Cyt-c (denoted as

state B1), with variable contributions of the reduced and oxi-

dized forms (Figure 1). The relative contributions of the com-

ponent spectra of reduced and oxidized forms obtained in this

way were converted to relative surface concentrations by

using proportionality factors determined by RR spectroscopy

of the pure species in solution.[43] The resultant potential-de-

pendent changes of the relative concentrations were then ana-

lyzed in terms of the Nernst equation for determining the stan-

dard reduction potentials E8. As summarized in Table 1, Cyt-c

electrostatically adsorbed to the different SAMs exhibits nearly

ideal Nernst behavior with E8values slightly more negative

than determined for Cyt-c in solution. These negative shifts

can be ascribed to the potential drop across the SAMs.[40]

The SERR spectra recorded at very negative potentials reveal

a small contribution (<5%) of native but redox-inactive spe-

cies which are attributed to proteins immobilized with orienta-

tions of very weak electronic coupling (Figure 2).[27,39] For elec-

trode potentials above 50 mV, the component analysis of the

SERR spectra reveals small contributions of the so-called B2

species of Cyt-c (Figure 2), in which the Met-80 axial ligand is

removed from the heme iron atom to give five-coordinate

high-spin heme species. This species is in equilibrium with a

non-native six-coordinate low-spin form in which the Met-80

ligand is replaced by a His residue.[3,40] This structural transition

to the B2 state is only observed for the ferric protein, whereas

upon reduction the B1 form is recovered.

Cyt-c was covalently attached to the various SAMs described

above by using two different

cross-linking reagents and proce-

dures (A and B; see Experimental

Section) that are expected to

yield differently oriented sam-

ples. In both cases the final step

consists of removal of the resid-

ual physisorbed Cyt-c by thor-

ough washing with buffered KCl

solutions. The desorption proce-

dure was optimized by monitor-

ing the SERR signal as a function

of time after dipping the Cyt-c-

loaded electrodes into KCl solu-

tions of different concentrations

(Figure 3). Accordingly, immersion of the electrodes in 2mKCl

solution for 15 min, followed by gentle rinsing with the buffer

solution, was found to remove 95% of the physisorbed species

from the electrode surface. This protocol was applied to the

electrode loaded with Cyt-c immobilized by covalent cross-link-

ing such that the contribution of electrostatically bound Cyt-c

to the resultant SERR spectra was always negligibly small.

Treatment with a sulfate-containing solution of the same ionic

strength did not cause complete desorption of the physisor-

bed proteins.

Figure 1. Soret-band SERR spectra measured at various potentials for Cyt-c

electrostatically adsorbed on a Ag electrode coated with a 1:1 C10-COOH/

C11-OH SAM. The experimental spectra are given by the black lines, whereas

the component spectra of the oxidized B1, the reduced B1, and the oxidized

B2 states are represented by the blue, red, and green lines, respectively.

Table 1. Thermodynamic and kinetic parameters obtained by SERR for Cyt-c in electrostatic and covalent com-

plexes with different SAMs. Kinetic constants for C10 and C5 SAMs refer to potential jumps from 41 to

200 mV and from 50 to 50 mV, respectively.

SAM Electrostatic binding Covalent binding[a]

C5-X C10-X C5-X C10-X

tail group

(X)

CO2HCO

2H/

CH2OH

CO2HCO

2H/

CH2OH

CO2HCO

2H/

CH2OH

CO2HCO

2H/

CH2OH

E0[mV] 1551932022126521535184

n0.8 0.9 0.9 0.9 0.6 0.5 0.6 0.5

kredox [s1][b] 22020 11310 16020 17020 12025 12510 12530 12020

bkorient [s1][c] 25025 17040 400100 25050 n.d.[d] n.d.[d] n.d.[d] n.d.[d]

[a] Covalent complexes prepared according to procedure A. [b] Determined by TRSERR with Soret-band excita-

tion. [c] Determined by TRSERR with Q-band excitation; [d] n.d.=not determined

ChemPhysChem 2010, 11, 1225 – 1235  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1227

Electron-Transfer Rates of Cytochrome c

Potential-dependent SERR spectra of Cyt-c covalently at-

tached to the various SAMs were measured in the potential

range from 400 to 50 mV, and their analysis in terms of differ-

ent contributions of the ferric and ferrous forms of state B1 in-

dicated that the chemical immobilization procedure does not

affect the structure of the redox center. However, about 10–

15% of the bound protein could not be reduced even at the

most negative potentials used (Figure 2). We ascribe this ob-

servation to the nonspecificity of the coupling reaction, which

in principle can lead to Cyt-c binding through any of the 11

lysine residues on the protein surface, so that a subpopulation

of protein molecules that are not able to establish efficient

electron pathways to the electrode results. Accordingly, Nernst

plots of covalently attached Cyt-c deviate from ideality with a

number of transferred electrons of 0.5–0.6 and slightly more

negative reduction potentials than observed for electrostatic

adsorption (Table 1 and Figure 2). As in the case of electrostatic

adsorption, SERR spectra indicate the formation of small

amounts of B2 species for potentials above 50 mV.

2.2. Orientation of the Immobilized Protein

For symmetric molecules, the surface-enhanced Raman (SER)

spectrum includes information about the orientation with re-

spect to the surface,[44] because for modes of different symme-

try the individual components of the scattering tensor are

modified to different extents depending on their relative orien-

tation with respect to the electric field vector. As a conse-

quence, vibrational modes of different symmetry may experi-

ence different enhancements. In the specific case of the heme

group which, in a first approximation, has D4hsymmetry, it can

be shown that the totally symmetric A1g modes will experience

preferential enhancement when the heme plane is parallel to

the surface, while for a perpendicular orientation the A1g

modes as well as the nontotally symmetric A2g,B

1g, and B2g

modes will be enhanced to the same extent.[30] Therefore, dif-

ferent orientations of the adsorbed heme protein are expected

to lead to different intensity ratios of modes of different sym-

metries, for example, n10(B1g)/n4(A1g). This is strictly true only

under nonresonant excitation conditions which, however,

would imply the loss of selectivity in probing the cofactor

spectrum. A reasonable compromise between an acceptable

molecular resonance enhancement and qualitatively predicta-

ble selection rules may be achieved upon excitation close to

the weak Q bands of the heme group. This approach was pre-

viously used to monitor orientational changes of Cyt-c electro-

statically adsorbed on electrodes coated with single-compo-

nent SAMs of w-carboxyl alkanethiols.[27,30] In that work, it was

shown that the orientation of the protein in the SAM/Cyt-c

electrostatic complexes is dependent on the electric field and

thus varies with the chain length of the w-carboxyl alkanethiol

and the applied electrode potential. Use of this method in the

present study indicated that dilution of the carboxyl-terminat-

ed thiols by addition of w-hydroxyl alkanethiols does not have

a significant impact on the average orientation of Cyt-c elec-

trostatically adsorbed on the SAMs. Both the absolute n10(B1g)/

n4(A1g) ratios and their dependence on potential are very simi-

lar to those of the pure carboxyl-terminated SAMs (Figure 4).

Similar to single-component SAMs,[27,30] the n10(B1g)/n4(A1g)

ratios decrease for the longer SAMs, which is consistent with

the proposed electric-field dependence of the orientation and

indicates a more perpendicular average orientation of the

Figure 3. Normalized SERR intensities of the n4band of reduced Cyt-c (B1) as

a function of the time of immersion in KCl solution of different concentra-

tions after immobilization by procedure A. Concentrations of KCl: 0.2m

(open squares), 1.0m(filled black triangles), 2.0m(open circles), 3.0m(open

triangles), saturated solution (filled black circles).

Figure 2. Relative concentrations of the different states of Cyt-c electrostati-

cally (top) and covalently bound (bottom) to C10-COOH/C11-OH SAMs, as

determined from Soret-band SERR spectra recorded as a function of the ap-

plied potential. The solid blue, red, green, and the open green symbols refer

to the oxidized B1, the reduced B1, the oxidized B2(6cLS), and the oxidized

B2(5cHS) states, respectively.

1228 www.chemphyschem.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2010, 11, 1225 – 1235

P. Hildebrandt, D. H. Murgida et al.

heme group with respect to the electrode surface for shorter

SAMs, that is, at higher electric fields.

Upon covalent binding of the protein to the SAMs by using

cross-linking procedure A, the n10(B1g)/n4(A1g) intensity ratio

drops with respect to the corresponding electrostatic com-

plexes by factors of about 2 and 1.3 for the shorter and longer

SAMs, respectively. These results indicate that, in the covalent

complexes, ferric Cyt-c is oriented with the heme group less

perpendicular to the surface than upon electrostatic binding.

At first sight, a different average orientation of the electrostati-

cally bound and the covalently attached Cyt-c may be surpris-

ing, since protocol A for covalent cross-linking is based on

electrostatic pre-adsorption of the protein to the SAM. Howev-

er, the lysine residues directly interacting with the carboxyl

functions of the SAM are less accessible for the cross-linker

EDC, such that amide bond formation may be favored for adja-

cent lysine residues. Furthermore, these steric constraints may

lead to a less homogeneous average orientation of the cova-

lently bound Cyt-c, which is consistent with the broad redox

transitions (see above). It appears that covalent attachment ac-

cording to procedure A lowers the stability of the protein.

When the electrode potential is set to E>E0, slow and partially

irreversible transition to the B2 or a B2-like state is observed

with time constants on the order of seconds or minutes even

for thick C11-SAMs (see Supporting Information, Figure S1).

Such a transition has not been noted for Cyt-c electrostatically

bound to SAMs of the same thickness. Covalent attachment of

Cyt-c by procedure A does not inhibit reorientation of the pro-

tein, as demonstrated by the dependence of the n10(B1g)/n4(A1g)

intensity ratio on potential, although the variation with poten-

tial is weaker than for electrostatically bound Cyt-c (Figure 5).

The situation is different when Cyt-c is covalently attached by

cross-linking procedure B. Here, the average orientation of the

heme group with respect to the electrode surface appears to

be even less perpendicular and, in contrast to covalent immo-

bilization by procedure A, it is independent of the applied po-

tential (Figure 5).

2.3. Electron Transfer and Orientation Dynamics

It is well established that the heterogeneous ET rate constant

of Cyt-c in electrostatic complexes with SAMs of long chain w-

carboxy alkanethiols exhibits the exponential dependence on

distance characteristic of the tunneling mechanism, but it

levels off when the number of methylene groups of the thiol is

reduced to less than ten.[2,3,23,31–36] This behavior is confirmed

by the present TRSERR experiments on electrostatically bound

Cyt-c carried out with an overpotential of 100 mV (Figure 6).

Figure 4. Q-band SERR spectra of Cyt-c in electrostatic complexes with C5-

COOH (black) and C5-COOH/C6-OH (gray) SAMs recorded at 0.1 (top) and

+0.1 V (bottom).

Figure 5. Potential dependence of the n10(B1g)/n4(A1g) intensity ratio as deter-

mined by Q-band SERR for oxidized Cyt-c immobilized on C10-COOH SAMs

by different strategies. Gray filled circles: electrostatic adsorption; black filled

circles: cross-linking procedure A; empty triangles: cross-linking procedure B.

Figure 6. Distance dependence of the apparent ET rate constants kapp

ET of

Cyt-c immobilized on C11-COOH SAMs, as determined by TRSERR spectros-

copy for overpotentials of 100 mV. Empty circles: electrostatically adsorbed

protein; empty triangles: protein cross-linked by procedure B.

ChemPhysChem 2010, 11, 1225 – 1235  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1229

Electron-Transfer Rates of Cytochrome c

The spectral analysis did not provide any indication for involve-

ment of species other than the reduced and oxidized form B1

in the interfacial redox process.[3,40,43] Essentially the same ki-

netic behavior is observed when the protein is electrostatically

adsorbed on mixed C10-COOH/C11-OH SAMs; the TRSERR ex-

periments yield very similar results (Table 1), in line with previ-

ous findings by Davis et al.[42] For the shorter SAMs, however,

we observe a drop of the apparent ET rate by a factor of two

when comparing C5-COOH with C5-COOH/C6-OH (Table 1). Q-

band-excited TRSERR spectra of electrostatically bound Cyt-c

reveal that, regardless of the SAM composition, thinner films

yield reorientation rates that are nearly identical to the ET rates

obtained from TRSERR experiments with Soret-band excitation.

These results are consistent with a gated mechanism, as re-

cently proposed for single-component SAMs,[2,27,30] in which

the overall redox process is controlled by the electron-tunnel-

ing probability at long distances, but protein reorientation be-

comes rate-limiting for the thinner films.

Covalent binding of Cyt-c to single-component or mixed

SAMs by cross-linking procedure A has a relatively small effect

on kapp

ET compared to the analogous electrostatic complexes

(Table 1), except for C5-COOH SAMs, for which a value nearly

two times smaller is determined. The overall similar kinetic be-

havior suggests that also for the covalent Cyt-c-SAM com-

plexes prepared according to procedure A the same gated

mechanism holds, even though the kinetics of the reorienta-

tion process could not be determined in an equally reliable

manner, mainly due to the smaller potential-dependent

changes of the n10(B1g)/n4(A1g) intensity ratio compared to the

electrostatic complexes (Figure 5). In this respect, the results

obtained for the covalently bound Cyt-c obtained by proce-

dure B are quite unexpected. Despite the lack of any detecta-

ble potential-dependent orientational changes, reflected by

the essentially constant n10(B1g)/n4(A1g) intensity ratio (Figure 5),

the kapp

ET values determined from the TRSERR experiments dis-

play qualitatively the same distance dependence as the elec-

trostatically adsorbed protein (Figure 6), albeit with generally

lower kapp

ET values. Also the overpotential dependence of kapp

for C10-COOH SAMs is qualitatively similar to that the electro-

static complexes (Figure 7), again with a limiting value that is

ca. 30% lower. When the relative viscosity of the solution is in-

creased to 1.5 cP by addition of sucrose, the kapp

ET values show

a decrease at the higher overpotentials which is more severe

than for the covalent complex obtained by procedure A. A fit

of Equation (2) to the experimental data for the overpotential

dependence of kapp

ET affords an apparent reorganization energy

of lapp =0.23 eV for Cyt-c electrostatically adsorbed to C10-

COOH SAMs. This value is in good agreement with that previ-

ously determined for Cyt-c at C15-COOH, for which electron

tunneling is the rate limiting step. This finding is consistent

with the fact that the reorientation rate is almost independent

of the applied overpotential (within the applied range) and

that kapp

reor is larger than the limiting value of kapp

ET (Figure 7). A

50% increase in solution viscosity causes a drop of the reorien-

tation rate of the electrostatically adsorbed protein that results

in a slight decrease of the kapp

ET values at the higher overpoten-

tials. As a consequence a slightly lower value of 0.19 eV is de-

termined for lapp. For the protein covalently bound by follow-

ing procedure B, the overpotential dependence of kapp

ET yields

lapp =0.20 eV at 1=1 cP, that is, a lower value than for the

electrostatic complex at 1 cP and very close to the value ob-

tained at 1.5 cP.

2.4. Molecular Dynamics Simulations

In previous computational work we studied the adsorption, dy-

namics, and electronic coupling of Cyt-c on metal surfaces

coated with SAMs of w-carboxyl alkanethiols.[27,39] It was shown

that the protein exhibits three main binding domains of differ-

ent affinity. Binding through any of the domains does not lead

to rigid electrostatic complexes, and instead the protein exhib-

its significant mobility that modulates the electronic coupling.

The simulations predict that small-amplitude motions of the

protein may have a substantial effect on the heterogeneous ET

rate. For instance, changes of the tilt angle of the heme plane

with respect to the electrode surface as small as 58may result

in a variation of the electronic coupling of one order of magni-

tude, corresponding to variations of kET by two orders of mag-

nitude. Furthermore, large-amplitude rotations of the protein

on the SAM surface are strongly suggested, although they

cannot be captured within the timescale of the simulations.

Such rotations have a direct impact on the order of magnitude

of the electronic coupling, while fine tuning is exerted by

Figure 7. Top: Overpotential dependence of kapp

ET for Cyt-c immobilized on

C10-COOH SAMs in normal phosphate buffer (1=1 cP; filled symbols) and

phosphate buffer with addition of sucrose (1=1.5 cP; empty symbols). Black

circles: electrostatically adsorbed protein; gray circles protein cross-linked by

method B. Bottom: Overpotential dependence of kapp

reor for C10-COOH/Cyt-c

electrostatic complexes at 1 cP (filled circles) and 1.5 cP (open circles).

1230 www.chemphyschem.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2010, 11, 1225 – 1235

P. Hildebrandt, D. H. Murgida et al.

structural thermal fluctuations of the proteins and solvent mol-

ecules.[27,39]

Herein, we applied the same methodology for investigating

the ET properties of Cyt-c covalently bound to similar SAMs.

For the in silico covalent linkage we selected as representative

examples four different lysine residues: lysines 79 and 86,

which are included in the main binding domains MZI and MZII

of the ferric protein, and lysines 13 and 72, which are included

in MZI and MZII of ferrous Cyt-c.[27,39] Single amide bonds with

the SAM were formed in each case. For all binding configura-

tions, MD simulations were run

in explicit water environment for

10 ns and optimal electronic

couplings Vmax were computed

every 10 ps by using the path-

way algorithm. Figure 8 shows

the mobility of the protein in

terms of the angles aand fthat

describe the orientation of the

heme group with respect to the

metal surface (see Experimental

Section for definition of aand f

and further computational de-

tails). The high-frequency mobili-

ty of the covalently attached protein is very similar to that ob-

served for electrostatic adsorption through the same region,

and thus leads to comparable fluctuations of the electronic

coupling along the simulation. Similarities and differences can

be better appreciated in the histograms shown in Figure 9. For

the angle a, the electrostatic and covalent complexes have

almost identical distributions centered at 1128, while for fthe

covalent complex is slightly shifted to lower values (maxima at

170 and 1728, respectively). Note that the dynamics of Cyt-c in

electrostatic and covalent complexes shown in Figure 8 are

rather similar during the first 8 ns. At this point in time the

electrostatically adsorbed protein rotates by 10 and 208in a

and fdirections, respectively, which is also evident as a

shoulder in Figure 9. The resulting orientation exhibits a lower

average coupling, and no large amplitude peaks are detected

for the remaining 2 ns. As a consequence, the ET rate in this

orientation is expected to be significantly lower. This large-am-

plitude rotation is impeded in the covalent complex. Similar re-

sults were obtained for all cases studied.

A summary of the results obtained for the different binding

configurations and redox states is presented in Table 2. In all

cases the adsorbed and covalently attached proteins exhibit

Figure 8. Variation of a(A), f(B), and V(C) as a function of simulation time

for oxidized Cyt-c cross-linked to a SAM through Lys86 (red) compared with

the variations of the same parameters for the equivalent electrostatic com-

plex (blue).

Figure 9. Histograms of the angles aand fdetermined along the 10 ns sim-

ulations for electrostatic (blue) and covalent (red) SAM/Cyt-c complexes.

Table 2. Average values of a,f, and V, maximum coupling VMAX and optimal electron pathways obtained for

the different immobilization modes of Cyt-c, as obtained by MD simulations.

Redox state Binding site <a><f>hVi[meV] VMAX [meV] ET path[a]

Covalent binding

Fe2+Lys13 93.8 172 0.140.075 0.61 Hec–Cys–SAM

Lys72 96 186 0.720.36 2.18 Hec–Cys–SAM

Fe3+Lys79 112 168 0.0950.046 0.52 Hec–Vinyl–SAM

Lys86 101 193 0.310.12 0.88 Hec–Vinyl–SAM

Electrostatic binding

Fe2+MZI (Lys13) 87 183 0.440.20 1.44 Hec-Cys-SAM

MZII (Lys72) 92 185 0.500.25 1.71 Hec–Cys–SAM

Fe3+MZI (Lys79) 113 171 0.0670.045 0.36 Hec–Wat–SAM

MZII (Lys86) 105 195 0.410.20 1.74 Hec–Methyl–SAM

[a] Hec: heme tetrapyrrole ring, Cys: cysteine 17, Methyl: methyl substituent of ring D, Wat: water molecule.

ChemPhysChem 2010, 11, 1225 – 1235  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1231

Electron-Transfer Rates of Cytochrome c

comparable average and maximum couplings as well as stan-

dard deviations.

2.5. Interplay of Electron Transfer and Protein Dynamics

The TRSERR results presented here show that the distance de-

pendence of the heterogeneous ET rate of Cyt-c on SAM-

coated electrodes follows qualitatively the same tendency

whether the protein is electrostatically adsorbed or cross-

linked to the SAMs. The kinetic behavior includes the charac-

teristic exponential variation for long SAMs and little or no var-

iation for the shorter ones. In quantitative terms, however, kapp

for the covalently attached protein is lower in the plateau

region. In all cases, kapp

ET , measured for short SAMs or high driv-

ing forces, drops upon increasing the viscosity of the solution

in contact with the immobilized protein, and this suggests that

under these conditions the rate-limiting event is related to pro-

tein motion. Stationary SERR experiments performed under Q-

band excitation show a distinct potential dependence of the

average protein orientation for electrostatically bound Cyt-c

and for covalent SAM/Cyt-c complexes prepared according to

procedure A, although in that case the variation is smaller. At

first sight this result is surprising, since formation of the amide

bond between the protein and the SAM is expected to restrict

the mobility. Although this is true for large-amplitude motion,

MD simulations show that the low-amplitude mobility (thermal

fluctuations) in the complexes formed through electrostatic in-

teractions and through equivalent single covalent bonds are

rather similar and, furthermore, that these subtle fluctuations

are sufficient for producing significant changes of the tunnel-

ing probabilities. Thus, the present experimental results for the

covalently bound Cyt-c prepared according to procedure A are

consistent with formation of a single amide bond that allows

for a similar orientation and low-amplitude mobility as in the

electrostatic complex. Small populations of differently oriented

protein or multibond formation cannot be discarded and, in

fact, are strongly suggested by the less ideal electrochemical

response and the slightly lower variation of the n10(B1g)/n4(A1g)

ratio with the potential in comparison with the electrostatic

complex. For electrostatically bound Cyt-c the transition from

the tunneling-controlled to the orientation-controlled ET

regime has been proposed to be governed by the increasing

interfacial electric field with decreasing SAM length, which

slows down the mobility of the protein. The experimentally de-

termined rate of reorientation of Cyt-c in electrostatic com-

plexes with C5-COOH SAMs is about two orders of magnitude

slower than that for C15-COOH SAMs,[30] corresponding to an

approximately twofold increase of the interfacial electric

field.[45] In agreement with this interpretation, MD simulations

on similar SAM/Cyt-c electrostatic complexes suggest that the

rate of reorientation from the high-affinity domain with low

electronic coupling configuration to the optimal orientation

for ET decreases by more than one order of magnitude in the

presence of an electric field strength of 0.01 V1and more

than 11 orders of magnitude when the field strength is

0.1 V1.[39] In view of the quite similar overall kinetic behavior

compared to electrostatically bound Cyt-c, we conclude that,

also for the covalent Cyt-c/SAM complex obtained by proce-

dure A, the local electric field strength determines the protein

mobility, despite partial neutralization of surface charges by

the cross-linking procedure.

According to the MD calculations, one would further expect

that, at sufficiently high fields, kapp

ET should decrease after reach-

ing a maximum. This behavior has not been observed for

SAMs of w-carboxyl alkanethiols, evidently because even for

the shortest possible chain length with only one methylene

group, the electric field is not sufficiently strong. A decrease of

kapp

ET , however, has in fact been observed upon replacing the

carboxyl group by divalent anions to increase the charge den-

sity at the interface and thus the electric field for the electro-

statically bound Cyt-c.[3] The distance dependence of kapp

ET for

Cyt-c covalently bound according to procedure B is qualitative-

ly similar to the two modes of protein immobilization dis-

cussed above (electrostatic and covalent through procedure A),

including the viscosity sensitivity in the plateau region. Howev-

er, SERR experiments performed with Q-band excitation do not

show any appreciable variation of the n10(B1g)/n4(A1g) ratio with

potential. This striking discrepancy indicates that procedures A

and B lead to different covalent Cyt-c/SAM complexes. Accord-

ing to protocol A, cross-linking is carried out with the electro-

static ferrous Cyt-c/SAM complex formed at a negative poten-

tial, while in protocol B the SAM is activated prior to coupling

of ferric Cyt-c at open circuit. Thus, procedures A and B yield

slightly different distributions of orientations, as reflected by

the different n10(B1g)/n4(A1g) intensity ratios. In addition, proto-

col B includes the addition of NHS and significantly longer in-

cubation times, and thus is expected to afford higher protein

coverage, as indicated by the stronger SERR signals. On the

other hand, albeit not verified experimentally, it is reasonable

to assume that protocol B should lead to the formation of

more than one amide bond per Cyt-c molecule.[42] If this were

the case the covalently attached protein would exhibit a much

more restricted mobility consistent with a potential-independ-

ent n10(B1g)/n4(A1g) intensity ratio. However, low-amplitude mo-

tions that refers to thermal fluctuations of the protein and in-

terfacial solvent molecules are not inhibited and may result in

a distinct modulation of the optimum electron pathways and

thus of the ET rate.[27,39] Moreover, depending on the positions

of the amide bonds the mobility of the protein can be restrict-

ed with respect to the angle a,f, or both. If the protein were

attached through two bonds that are contained in a plane per-

pendicular to the heme group, then rotation with respect to a,

but not to f, would be largely restricted. As a consequence,

SERR experiments would reveal no changes of the n10(B1g)/

n4(A1g) intensity ratio that are only sensitive to variation of a.

Conversely, thermal fluctuations and low-amplitude rotations

with respect to fthat are not detectable in SERR experiments

may still occur and modulate the electronic coupling. Then, as

in the previous cases, protein dynamics is expected to be rate-

limiting for thin SAMs, that is, at high electric fields, as sug-

gested by the large viscosity effects observed for short SAMs

or high driving forces.

Consistent with this interpretation are previous findings by

Jin et al., who showed that, in contrast to electrostatically im-

1232 www.chemphyschem.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPhysChem 2010, 11, 1225 – 1235

P. Hildebrandt, D. H. Murgida et al.

mobilized Cyt-c, the covalently bound protein prepared ac-

cording to procedure A is not capable of shuttling electrons

between the electrode and NADPH cytochrome P450 reduc-

tase in the solution phase.[46] Here a major reorientation is re-

quired to switch between electron exchange with the elec-

trode and the reductase, a movement of Cyt-c that is blocked

upon covalent attachment already under “mild” conditions.

Conclusions

The results presented here show that the experimentally deter-

mined rate constants for the heterogeneous ET of Cyt-c immo-

bilized on SAM-coated electrodes represent a convolution of

orientation-dependent tunneling probabilities and the orienta-

tional distribution and dynamics of the protein ensemble. In

electrostatic complexes with negatively charged SAMs, the rel-

atively narrow orientational distribution leaves the vast majori-

ty of molecules in an orientation that is not optimized for ET. A

qualitatively similar conclusion is reached for the protein cross-

linked to these SAMs, although in this case the distribution of

orientations is broader and the mobility is restricted specifically

when the protein is attached by more than one covalent

amide bond. However, even in the covalent complexes, low-

amplitude motions of the proteins and interfacial water mole-

cules guarantee transient pathways of sufficiently high elec-

tronic coupling. For thick SAMs, electron tunneling is signifi-

cantly slower than protein and solvent dynamics, and thus the

measured rates represent a true ET rate that exhibits the char-

acteristic exponential dependence on distance. For thinner

SAMs, that is, higher charge densities and stronger electric

fields, protein and solvent dynamics is significantly slowed

down and becomes rate-limiting. This kinetic behavior is likely

to be rather general and may constitute the basis for the un-

usual distance dependencies of the ET rates reported for a vari-

ety of redox proteins in electrochemical studies.[21–26] A similar

mechanism is likely to operate when Cyt-c exerts its electron-

transport function in aerobic respiration. As suggested by vari-

ous studies, the initial electrostatic complex of Cyt-c with the

natural reaction partner Cyt-c oxidase is not optimized for ET

and thus requires reorientation.[47] According to the results pre-

sented here, such reorientation processes can be expected to

be modulated by the variable transmembrane potential, which

thus would play a role in inhibitory control of the ET-driven

proton translocation activity.

Experimental Section

Chemicals: 6-Mercaptohexanoic acid (C5-COOH) was purchased

from Dojindo. All other chemicals, including 11-mercaptoundeca-

noic acid (C10-COOH), 11-mercaptoundecan-1-ol (C11-OH), 6-mer-

captohexan-1-ol (C6-OH), N-ethyl-N’-(3-dimethylaminopropyl) car-

bodiimide (EDC), N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide

methyl-p-toluenesulfonate (CMC), and N-hydroxysuccinimide (NHS),

were purchased from Sigma-Aldrich and used without further pu-

rification. Horse heart cytochrome c (Cyt-c) was purchased from

Sigma-Aldrich and purified by HPLC. The water used in all experi-

ments was purified by a Millipore system and its resistance was

greater than 18 MW.

Electrode Modification: Silver ring electrodes were mechanically

polished with 3M polishing films from 30 to 1 mm grade. After

washing, electrodes were subjected to oxidation–reduction cycles

in 0.1mKCl to create an SER-active nanostructured surface. Subse-

quently, the electrodes were incubated in 1.5 mmethanolic solu-

tions of the alkanethiols (pure or 1:1 mixtures) for ca. 20 h, and

then rinsed and transferred to the spectroelectrochemical cell. For

electrostatic adsorption Cyt-c was added to the electrochemical

cell from a stock solution to form a 0.2–0.4 mmsolution and al-

lowed to incubate at room temperature for ca. 15–20 min before

starting the experiments.

Covalent Binding: Two different procedures were used for cross-

linking of Cyt-c to the SAMs. In procedure A, the protein was ad-

sorbed on the SAM-coated working electrode for about 20 min. at

100 mV as in the case of electrostatic binding. Subsequently, EDC

was added to the cell from a stock solution to obtain a 5 mmcon-

centration. The resulting solution was stirred for 45 min while

maintaining the applied potential (100 mV). The electrode was

then rinsed with water and exposed to a 2–3mKCl solution for

15 min at open circuit to remove remaining electrostatically ad-

sorbed protein.

In procedure B, The SAM-coated electrodes were incubated for 2 h

in a de-aerated solution of CMC (20 mg/10 mL) and NHS (6 mg/

10 mL). After activation of the carboxyl groups, the electrode was

rinsed thoroughly and immersed in a 10 mmCyt-c solution con-

taining 30mmphosphate buffer solution (pH 7) overnight. Finally,

the electrode was thoroughly rinsed with water and then im-

mersed in a 2–3mKCl solution for 10 min to desorb the remaining

physisorbed protein. In contrast to procedure A, all steps were per-

formed at open circuit.

Surface-Enhanced Resonance Raman Spectroscopy: The spectro-

electrochemical cell for SERR spectrosocpy has been described

elsewhere.[40] Briefly, a Pt wire and a Ag/AgCl electrode were used

as counter- and reference electrodes, respectively. All potentials

cited in this work refer to the Ag/AgCl (3mKCl) electrode. The

working electrode was a silver ring of 8 mm diameter and 2.5 mm

height mounted on a shaft that is rotated at about 5 Hz to avoid

laser-induced sample degradation.

The electrolyte solution (30 mmphosphate buffer, pH 7.0) was

bubbled with catalytically purified oxygen-free argon prior to the

measurements, and Ar overpressure was maintained throughout

the experiments. The viscosity of the solution was adjusted by ad-

dition of sucrose to the same buffer, which has been shown not to

affect significantly the dielectric constant.[33]

SERR spectra were measured in backscattering geometry by using

a confocal microscope coupled to a single-stage spectrograph

(Jobin Yvon, LabRam 800 HR or XY 800) equipped with a liquid-ni-

trogen-cooled back-illuminated CCD detector. Elastic scattering

was rejected with notch or edge filters. The 413 nm line of a cw

krypton ion laser (Coherent Innova 300c) or the 514 nm line of a

cw argon laser (Coherent Innova 70c) was focused onto the surface

of the rotating Ag electrode by means of a long-working-distance

objective (20, N.A. 0.35). Typically, experiments were performed

with laser powers of about 1 mW (413 nm) and 5–12 mW (514 nm)

at the sample. Effective acquisition times were between 3 and 10 s.

All experiments were repeated several times to ensure reproduci-

bility.

For TRSERR experiments, potential jumps of variable height and

duration were applied to trigger the reaction. The SERR spectra

were measured at different delay times following the potential

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Electron-Transfer Rates of Cytochrome c

jump. Synchronization of potential jumps and probe laser pulses

was achieved by a pulse-delay generator (BNC). The probe pulses

were generated by passing the cw laser beam through two con-

secutive laser intensity modulators (Linos), which give a total ex-

tinction better than 1:50000 and a time response of ca. 20 ns. De-

tails of the TRSERR measurements are described elsewhere.[23,30]

After background subtraction the spectra were treated by single-

band (514 nm) or component analysis[43] (413 nm) in which the

spectra of the individual species were fitted to the measured spec-

tra by using a home-made analysis software. The time-dependent

changes of the relative concentrations of the species involved

were subsequently analyzed in terms of relaxation kinetics to yield

reciprocal relaxation time constants.[24]

Molecular Dynamics Simulations: The computational methods for

comparing the covalent and electrostatic SAM/Cyt-c complexes

have been presented and discussed previously,[27,39] and therefore

they will be only briefly described here.

Initial Cyt-c structures were obtained from the PDB database (PDB

26IW and PDB 1HRC for the reduced and oxidized form, respective-

ly). For simulating the SAMs, an infinite array of fixed Au atoms

with lattice structure 111 was built in silico, and each of them was

linked to a C5-COOH molecule through the S atom. SAM and lat-

tice parameters were adopted from the literature.[27,39] The infinite

array consisted of a periodic boundary condition (PBC) cell of

1314 Au atoms in the Xand Ydirections, with their correspond-

ing alkanethiols. In the Zdirection periodicity is achieved by using

periodic boxes of 80  width, which leaves sufficient space be-

tween the Cyt-c surface and the next (upper) gold layer to avoid

direct interaction. Cross-linking of Cyt-c to the SAMs was per-

formed by in silico modification of selected protein Lys residues

and SAM carboxylate groups to an amide bond.

The production simulations of each complex were performed by

immersing the SAM/Cyt-c structures contained in a TIP3P water

box. For each case, an initial constant-volume MD was performed

to heat the system to 300 K, and subsequently a constant-pressure

simulation was performed to equilibrate the system density. Finally,

production MDs were performed. Temperature and pressure were

kept constant using the Berendsen thermostat and barostat. For

the PBC simulations, Ewald summations were employed to com-

pute the electrostatic energy terms by using the default parame-

ters in the Sander module of the AMBER package.

The orientation of Cyt-c with respect to the Au/SAM surface was

defined based on the relative heme orientation in terms of two

angles: the angle abetween the FeS(Met80) bond, which is per-

pendicular to the heme plane, and the Zaxis of the system, which

is perpendicular to the Au/SAM surface. Values of aclose to 0 or

1808imply that the heme group lies parallel to the SAM, whereas

a value of 908corresponds to perpendicular orientation of the

heme plane with respect to the surface. The angle fis determined

by the vector defining the FeNAbond (where NAis the nitrogen

atom of the pyrrolic ring) and the vector pointing towards the

SAM, which lies in the heme plane. This angle describes the rota-

tional orientation of the heme group and thus of the entire Cyt-c.

Values between 0 and 908correspond to protein orientations in

which the heme propionate groups are closer to the SAM surface,

whereas for values between 180 and 2708the propionate groups

point away from the SAM surface. Note that for avalues close to 0

or to 1808changes of the fvalue do not correspond to significant

variations of protein orientation since the heme group lies parallel

to the SAM surface.

Coupling Matrix calculation: The electronic coupling between the

heme iron atom and any of the Au atoms that represent the elec-

trode surface were estimated by using the pathway algorithm de-

veloped by Beratan et al.,[48] specifically modified for the present

system.[27,39]

Acknowledgements

Financial support by the DFG (Sfb498-A8; PH, D.H.M.), the Fond

der Chemischen Industrie (I.M.W.) and the ANPCyT (PICT2006-

459; PICT2007-00314) is gratefully acknowledged. M.A.M. and

D.H.M. are members of CIC-CONICET. D.F.M. and D.A.P. are CONI-

CET fellows.

Keywords: electron transfer ·molecular dynamics ·

monolayers ·proteins ·time-resolved spectroscopy

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Received: December 8, 2009

Published online on April 7, 2010

ChemPhysChem 2010, 11, 1225 – 1235  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 1235

Electron-Transfer Rates of Cytochrome c

Electric Field Effects on the Interfacial Electron Transfer

and Protein Dynamics of Cytochrome c

Ly, H. K.; Wisitruangsakul, N.; Sezer, M.; Feng, J. J.; Kranich, A.; Weidinger, I.; Zebger,

I.; Murgida, D. H.; Hildebrandt, P. J. Electroanal. Chem. 2011, 660, 367-376.

Electric-ﬁeld effects on the interfacial electron transfer and protein dynamics

of cytochrome c

H. Khoa Ly

, Nattawadee Wisitruangsakul

a,b

, Murat Sezer

, Jiu-Ju Feng

a,c

, Anja Kranich

Inez M. Weidinger

, Ingo Zebger

, Daniel H. Murgida

, Peter Hildebrandt

⇑

Technische Universität Berlin, Institut für Chemie, Sekr. PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany

Iron and Steel Institute of Thailand, 1st-2nd Floor, Bureau of Industrial Sectors Development Building, Trimitr Soi., Pharam 4 Rd. Phakhanong, Klongtoey Bangkok 10110, Thailand

School of Chemistry and Environmental Science, Henan Normal University, Xinxiang, Henan 453007, China

Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,

Pab. 2, Piso. 1, C1428EHA-Buenos Aires, Argentina

article info

Article history:

Received 18 May 2010

Received in revised form 8 October 2010

Accepted 15 December 2010

Available online 21 December 2010

Keywords:

Surface enhanced Raman spectroscopy

Surface enhanced infrared spectroscopy

Electron transfer

Cytochrome c

Electric ﬁeld

abstract

Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectros-

copy have been employed to study the interfacial redox process of cytochrome c(Cyt-c) immobilised

on various metal electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated mer-

captanes. The experiments, carried out with Ag, Au and layered Au–SAM–Ag electrodes, afford apparent

heterogeneous electron transfer constants (k

relax

) that reﬂect the interplay between electron tunnelling,

redox-linked protein structural changes, protein re-orientation, and hydrogen bond re-arrangements in

the protein and in the protein/SAM interface. It is shown that the individual processes are affected by

the interfacial electric ﬁeld strength that increases with decreasing thickness of the SAM and increasing

difference between the actual potential and the potential of zero-charge. At thick SAMs of mercaptanes

including 15 methylene groups, electron tunnelling (k

) is the rate-limiting step. Pronounced differences

for k

and its overpotential-dependence are observed for the three metal electrodes and can be attrib-

uted to the different electric-ﬁeld effects on the free-energy term controlling the tunnelling rate. With

decreasing SAM thickness, electron tunnelling increases whereas protein dynamics is slowed down such

that for SAMs including less than 10 methylene groups, protein re-orientation becomes rate-limiting, as

reﬂected by the viscosity dependence of k

relax

. Upon decreasing the SAM thickness from 5 to 1 methylene

group, an additional H/D kinetic isotope effect is detected indicating that at very high electric ﬁelds re-

arrangements of the interfacial or intra-protein hydrogen bond networks limit the rate of the overall

redox process.

1. Introduction

Interfacial electron transfer (ET) reactions play a key role in

various processes of technological importance such as catalysis,

corrosion and energy conversion or storage [1–3]. Furthermore,

they are constitutive for a large number of biological functions

and essential for most of the biotechnological applications that

utilize redox enzymes [4]. This wide range of fundamental and

applied aspects of heterogeneous ET has motivated numerous

experimental and theoretical studies for decades. It is, therefore,

quite surprising that these elementary reactions are yet not com-

prehensively understood.

One reason for this quite remarkable gap is related to method-

ological shortcomings. For a long time, electrochemical methods

have been the only techniques for determining thermodynamic

and kinetic data of redox processes although the nature of the par-

ticipating molecules could not be directly identiﬁed [5,6]. Thus,

analyses of mechanistic aspects of interfacial redox processes had

to rely upon indirect evidences.

In this respect, surface enhanced Raman (SER) and surface en-

hanced infrared absorption (SEIRA) spectroscopy represent power-

ful complementary techniques since the vibrational spectra of

molecules in close vicinity of metal electrodes can be selectively

probed due to the resonant coupling of the radiation ﬁeld with

the surface plasmons of the metallic support, which may be Ag

or Au [7,8]. These methods can be coupled with the potential jump

technique such that they provide information of the kinetics and

thermodynamics of the interfacial processes and the nature of

the molecule species involved [9–13].

SER and SEIRA spectroscopy have been employed to study inter-

facial processes of redox proteins [14–17]. In these studies, the SER

doi:10.1016/j.jelechem.2010.12.020

⇑

Corresponding author. Tel.: +49 30 314 21419; fax: +49 30 314 21122.

E-mail address: [email protected] (P. Hildebrandt).

Journal of Electroanalytical Chemistry 660 (2011) 367–376

Contents lists available at ScienceDirect

Journal of Electroanalytical Chemistry

journal homepage: www.elsevier.com/locate/jelechem

effect is combined with the molecular resonance Raman (RR) effect

(surface enhanced resonance Raman – SERR) such that it is possible

to selectively probe the redox site solely of the immobilised pro-

teins by tuning the excitation line in resonance with both the elec-

tronic transition of the cofactor and the surface plasmons of the

metal [7,8]. The sensitivity and selectivity of SEIRA spectroscopy

is increased upon operating in the difference mode such that only

potential-dependent changes of the vibrational bands of the pro-

tein and the cofactor are detected [13,16,17].

A widely used model system, appropriate for employing SERR

and SEIRA spectroscopy as well as electrochemical methods, is

based on Au or Ag electrodes coated by self-assembled monolayers

(SAM) of mercaptanes [14,15]. Such coatings on Au surfaces have

been ﬁrst characterised by Nuzzo and Allara [18] and later used

for biocompatible immobilisation of redox proteins [19–21]. The

terminal tail groups of the mercaptanes can be varied to allow

for different modes of protein binding and the thickness of the

SAM is deﬁned by the number of methylene groups in the alkyl

chain of the mercaptanes. These devices allow systematic variation

of important parameters controlling the heterogeneous ET, such as

the ET distance, driving force, charge distribution in the SAM/pro-

tein interface, and ionic strength, viscosity, and pH in the bulk

solution.

A particularly large body of experimental and theoretical data

has been accumulated for the redox process of the heme protein

cytochrome c(Cyt-c) electrostatically immobilised on electrodes

coated with carboxyl-terminated SAMs [11–17,20–50]. The results

obtained so far indicate that the overall redox process is deter-

mined by the coupling of protein dynamics with electron tunnel-

ling [42,46–48]. This coupling results from the fact that the

energetically preferred electrostatic binding domain corresponds

to an orientation of the bound protein that exhibits a distinctly

lower tunnelling probability than a lower afﬁnity binding site

[46,48]. Optimum electron tunnelling efﬁciency, therefore, re-

quires rotational motions of the protein on the SAM. This conclu-

sion seems to provide a satisfactory explanation for the unique

distance-dependence of the experimentally observed ET rate

which ﬁrst increases exponentially with decreasing distance, i.e.

the SAM thickness, but then levels off to a plateau region for SAMs

shorter than 10 methylene groups [12–15,23,25,27,29–32,42]. This

region has, hence, been attributed to a kinetic regime in which pro-

tein-re-orientation becomes the rate-limiting step [42]. This inter-

pretation is consistent with the experimental ﬁnding that the

relaxation constants for protein re-orientation decrease substan-

tially with decreasing distance in contrast to the exponential in-

crease of the electron tunnelling rate. The slow-down of the

protein dynamics with decreasing distance has been attributed to

the concomitant increase of the electric ﬁeld strength at Cyt-c/

SAM interface, thereby increasing the activation energy for protein

re-orientation. Thus, the interfacial redox process in toto has been

suggested to be modulated by the electric ﬁeld. Evidently, this sce-

nario holds for quite different proteins which have been shown to

exhibit a similar non-exponential distance dependence of the het-

erogeneous ET rate constant [51–55].

However, there are still some observations which do not fully ﬁt

into this scheme. First, in the limit of highly restricted protein

mobility, one would expect at least a slight increase of the ET rate

with further decreasing the electron tunnelling distance due to the

exponential increase of the electronic coupling parameter in each

protein orientation. However, all experimental data reported so

far indicate an essentially distance-independent regime for short

distances, i.e. high electric ﬁelds. Second, although experimental

data obtained for SAM-coated Au and Ag electrodes display a qual-

itatively similar distance dependence, even for the same protein

(i.e. Cyt-c) the absolute values for the experimentally determined

ET rate constants are generally higher for Au than for Ag

[12,13,23,25,27–29,36,38,45]. Third, in the plateau region, the ET

rate constants have been shown to be lower in D

O compared to

O, which has been ascribed to a kinetic isotope effect due to

the coupling of electron transfer with redox-linked proton translo-

cation in the protein or in the protein/SAM interface [12,43].

Although recently a part of this isotope effect was attributed to

the intrinsically higher viscosity of D

O solutions [43], it remains

to be clariﬁed why the ratio of the rate constants in H

O and D

increases with decreasing distance to the electrode.

In the present work we have addressed these three questions by

employing time-resolved (TR) SERR and SEIRA spectroscopy to

probe the distance- and overpotential-dependence of the interfa-

cial ET on Ag, Au, and Au–SAM–Ag hybrid electrodes. Special

emphasis is laid on the analysis of viscosity and H/D effects on

the kinetic constants. The results lead to the conclusion that not

only protein re-orientation but also the electron tunnelling step it-

self is controlled by the interfacial electric ﬁeld.

2. Experimental

2.1. Materials

6-mercaptohexanoic acid (C

) from Dojindo, 16-mercaptohexa-

decanoic acid (C

), 11-mercaptoundecanoic acid (C

), and mer-

captoacetic acid (C

), all purchased from Sigma–Aldrich, were

used without further puriﬁcation. Formation of SAMs on the metal

electrode followed the protocol described previously [26]. Horse

heart Cyt-c from Sigma–Aldrich was puriﬁed by HPLC. The water

used in all experiments was puriﬁed by a Millipore system and

its resistance was more than 18 M

. All other chemicals were of

highest purity grade available.

2.2. Surface Enhanced Resonance Raman spectroscopy

The spectroelectrochemical cell for SERR spectrosocpy has been

described elsewhere [26]. All potentials cited in this work refer to

the Ag/AgCl (3 M KCl) electrode. A rotating Ag ring served as the

working electrode. SER-activation, coating of the electrode by

SAMs and subsequent protein immobilization followed the proto-

col described previously with minor modiﬁcations [10,26]. Brieﬂy,

prior to the spectroscopic experiments, the electrolyte solution

(30 mM phosphate buffer, pH = 7.0) was bubbled with catalytically

puriﬁed oxygen-free argon for ca. 20 min. Consecutively, Cyt-c

solution was added yielding a ﬁnal concentration of ca. 0.2

Protein adsorption was achieved by incubating the working elec-

trode for 30 min into the protein containing buffer solution at open

circuit potential. All experiments were performed in the presence

of protein in solution and Ar overpressure.

For experiments in D

O, the protein was dissolved in buffered

O solutions adjusted to pD = 7.0 [12] and subsequently incu-

bated for further 18–24 h for complete H/D exchange. To account

for the intrinsically higher viscosity of D

O, comparative experi-

ments in H

O buffer were carried out at a viscosity of 1.2 cp

adjusted by addition of sucrose [42]. All SERR measurements were

carried out with a roughened Ag electrode [10] or an Au–SAM–Ag

electrode fabricated as described previously [45].

SERR spectra were measured in back-scattering geometry using

a confocal microscope coupled to a single stage spectrograph (Jobin

Yvon, LabRam 800 HR) equipped with a liquid-nitrogen cooled

back illuminated CCD detector. The 413-nm line of a cw Krypton

ion laser (Coherent Innova 300c) was focused onto the surface of

the rotating Ag electrode by means of a long working distance

objective (20; NA 0.35). Typically, experiments were performed

with laser powers of ca. 1 mW. Effective acquisition times were be-

tween 3 and 10 s. All experiments were repeated several times to

368 H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376

ensure reproducibility. For TR-SERR experiments, potential jumps

of variable height and duration were applied to trigger the

reaction. The SERR spectra were measured at different delay times

following the potential jump. Further details of the stationary and

TR-SERR experiments are given elsewhere [11,15,27]. After back-

ground subtraction the spectra were treated by a component analy-

sis in which the spectra of the individual species were ﬁtted to the

measured spectra using a home-made analysis software [56,57].

2.3. Surface enhanced infrared absorption measurements

SEIRA measurements were performed with a Kretschmann-ATR

conﬁguration using a semi-cylindrical shaped silicon crystal. Thin

gold (Au) ﬁlms were formed on the ﬂat surface of the silicon

substrate by electroless (chemical) deposition technique [13]. For

Cyt-c adsorption, SAM-coated electrodes were immersed in Cyt-c

solution using a concentration of 2

M. The SEIRA spectra were

recorded from 4000 to 1000 cm

1

with a spectral resolution of

4cm

1

on a Bruker IFS66v/s spectrometer equipped with a photo-

conductive MCT detector. Four hundred scans were co-added for a

spectrum. For the spectro-electrochemical measurements, the ATR

crystal was incorporated into a three-electrode home-built cell

[13].

TR SEIRA experiments were carried out using the potential

jump technique [13]. Spectra acquisition was synchronized with

the potential jumps controlled by a home-made pulse delay gener-

ator. Series of time dependent single-channel spectra of Cyt-c were

collected when a potential jump was carried out from the reference

potential at which Cyt-c was either largely reduced (0.1 V) or oxi-

dised (+0.125 V) to the ﬁnal potential, and for the reverse jump.

Depending on the time scale under examination, either step-scan

or rapid-scan TR SEIRA measurements were carried out. The photo-

conductive MCT detector with a fast ampliﬁer was used in the

DC-coupled mode for the step-scan measurements. In the case of

the rapid-scan measurements, the AC-coupled mode was utilized.

Details of the set-up as well as of the stationary and TR SEIRA

experiments have been described previously [13].

3. Results

3.1. Stationary SERR spectra and spectra analysis

SERR spectra of Cyt-c were measured from Ag electrodes coated

with carboxyl-terminated thiols of different chain lengths

-SAM), expressed by the number of methylene groups x. For

chain lengths with x= 15, 10, 5, SERR spectra do not provide any

indication for contributions from species other than the native

state (denoted as B1). Thus, these spectra could be well described

by a superposition of the reduced and oxidised forms of state B1

using component analysis. For shorter SAMs, however, there are

non-negligible contributions from non-native species (denoted as

state B2) which increase upon approaching potentials above the

redox potential of the native state [57,58], as reﬂected, inter alia,

by a broadening of the peaks in the

and

band region

(Fig. 1). These species include a ﬁve-coordinated high spin (5cHS)

and a six-coordinated low spin (6cLS) ferric heme in which the

native Met80 ligand is removed from the heme iron and this

coordination site remains vacant or is replaced by a His, respec-

tively [57]. The formation of these species is most likely related

to the weakening of the Fe-Met80 bond of native ferric Cyt-c

(B1) by the high electric ﬁelds in close proximity to the electrode

as concluded from previous experimental and theoretical studies

[57–59]. Thus, the analysis of the SERR spectra measured under

these conditions has to consider the involvement of four different

species, i.e., B1

red

,B1

,B2

[5cHS], and B2

[6cLS]. Then, the

component analysis provides a consistent description of all exper-

imental spectra, yielding the relative spectral contributions of each

component to the individual experimental spectra. These spectral

contributions were subsequently converted to relative concentra-

tions [57]. The results of the analysis of the SERR spectra of Cyt-c

at a C

-SAM-coated Ag electrode, measured as a function of the

electrode potential, are shown in Fig. 2. The only redox transition

refers to the native state B1 as demonstrated by a ﬁt of the Nerns-

tian equation to the data, allowing for the determination of the re-

dox potential E

and the number of transferred electrons n

(Table 1). No effect on the redox equilibrium was observed upon

increasing the viscosity of the solution or upon H/D exchange.

The non-native species are only present in the oxidised forms.

The corresponding reduced counterparts are formed with electrode

potentials below 0.3 V and are then rapidly converted to the na-

tive reduced B1 state [58,60], such that they are not detectable un-

der equilibrium conditions in the stationary measurements.

3.2. Distance-dependence of the apparent electron transfer rate

constant

TR-SERR experiments were ﬁrst performed employing potential

jumps from negative potentials (0.1 V) to the redox potential

such that relaxation processes include the ET of the immobilised

Cyt-c at zero-driving force (

G= 0 eV). These experiments were

carried out as a function of the SAM thickness for x= 15, 10, 5,

and 1 on Ag electrodes. As in the stationary experiments, spectra

for xP5 only include the native species whereas at x= 1 also

the component spectra of the two B2 species were required for a

Fig. 1. SERR spectra of Cyt-c immobilised on a C

-SAM-coated Ag electrode at

different electrode potentials. The blue, red, dark gray, and light gray lines represent

the component spectra of the reduced B1, oxidised B1, oxidised B2[6cLS], and

oxidised B2[5cHS] state, respectively. Further experimental details are given in the

text. (For interpretation of the references to colour in this ﬁgure legend, the reader

is referred to the web version of this article.)

H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376 369

satisfactory global ﬁt to the experimental TR SERR spectra. Repre-

sentative example spectra of experiments with Cyt-c on a C

SAM-coated Ag electrode are shown in Fig. 3. The time-dependent

relative concentration changes of the various species indicate a

clear correlation between the decay of the reduced and the

increase of the oxidised B1 state (Fig. 4). The non-native species

initially grow in and then reach a constant level after ca. 20 ms.

Thus, the kinetic analysis cannot be restricted to the redox pro-

cess of the native state but must also include the formation of

the B2 species. A qualitatively similar kinetic behaviour has been

recently observed for Cyt-c covalently attached to SAM-coated Ag

electrodes, and found to be adequately described by the scheme

in which B1

exhibits a reaction channel to B2

[5cHS] in

equilibrium with B2

[6cLS] [47]. In a similar way, we have

considered the temporal evolution of the B2 species in the

present kinetic analysis which thus allowed determining the

relaxation constant k

relax

of the redox process of B1 (Table 1).

For the C

-SAM, k

relax

was identical to the previously deter-

mined value [12] but with decreasing chain length the relaxation

constants obtained in this work were found to be smaller than

the constants reported earlier, speciﬁcally for the C

-SAM. As a

consequence, the distance-dependence of k

relax

does not display a

plateau for x< 10 but even a decrease at x<5(Fig. 5).

Table 1

Redox potentials and relaxation constants at zero-driving force for the interfacial

redox process of Cyt-c immobilised on Ag electrodes coated with different SAMs.

SAM Buffer E

(V) k

relax

1

)

O 0.021 53

O, 1.2 cp 0.019 45

O 0.020 48

O 0.018 130

O, 1.2 cp 0.010 100

O 0.015 100

O 0.033 40

O, 1.2 cp 0.031 30

O 0.030 16

The data were obtained by TR-SERR experiments with potential jumps from

0.1 V to the redox potential. The average error associated with the determination

of E

and k

relax

was determined to be ±005 V and ±10%.

Fig. 3. Selection of SERR spectra of a time-resolved experiment of Cyt-c immobi-

lised on a C

-SAM-coated Ag electrode, including the stationary SERR spectra at the

initial potential E

=0.1 V and the ﬁnal potential E

, and a TR SERR spectrum

obtained after a delay time d= 18 ms subsequent to the potential jump. The blue,

red, dark gray, and light gray lines represent the component spectra of the reduced

B1, oxidised B1, oxidised B2[6cLS], and oxidised B2[5cHS] state, respectively.

Further experimental details are given in the text. (For interpretation of the

references to colour in this ﬁgure legend, the reader is referred to the web version of

this article.)

Fig. 4. Time-dependent changes of the relative concentrations of the various

species of Cyt-c immobilised on a C

-SAM-coated Ag electrode, following a potential

jump from E

=0.1 V to the E

. The experimental data were determined by TR

SERR spectroscopy. The blue, red, dark gray, and light gray symbols represent the

reduced B1, oxidised B1, oxidised B2[6cLS], and oxidised B2[5cHS] state, respec-

tively. The solid lines refer to a ﬁt of exponentials to the experimental data. Further

details of the experiments and the data analysis are given in the text. (For

interpretation of the references to colour in this ﬁgure legend, the reader is referred

to the web version of this article.)

Fig. 2. Potential-dependent distribution of the relative concentrations of the various

species of Cyt-c immobilised on a C

-SAM-coated Ag electrode, as determined by

SERR spectroscopy. The blue(circles), red(squares), dark gray(down-pointing trian-

gle), and light gray(up-pointing triangle) symbols represent the reduced B1, oxidised

B1, oxidised B2[6cLS], and oxidised B2[5cHS] state, respectively. The solid lines refer

to a ﬁt of the Nernst equation to the experimental data. Further experimental details

are given in the text. (For interpretation of the references to colour in this ﬁgure

legend, the reader is referred to the web version of this article.)

370 H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376

3.3. Kinetic isotope and viscosity effects of the apparent electron

transfer rate constant

Preliminary SEIRA experiments monitoring the amide I band

changes indicated that a complete H/D exchange requires incuba-

tion times for more than 24 h which is consistent with previous

studies revealing a wide dynamic range for the substitution of

the exchangeable protons [61,62]. Here we have chosen two differ-

ent protocols for the H/D exchange. First, ferric Cyt-c was incu-

bated in D

O buffer solutions (pD 7.0) for different periods of

time from 1 h to 24 h. Second, to achieve an exchange of protons

that are hardly accessible in the folded form, the protein was ﬁrst

unfolded in the presence of GuHCl at pH 2.0 in D

O and subse-

quently refolded in neutral (buffered) D

O solution [63]. However,

in all cases, the kinetic constants, determined for a potential jump

to the redox potential at a given SAM by SERR spectroscopy, were

found to be the same.

TR-SERR experiments on Ag electrodes coated with SAMs with

xP10 in D

O afforded relaxation constants equal to those

determined in H

O whereas for C

-SAM-coated Ag electrode, the

experiments yielded a 1.3 times smaller value in D

O(Table 1). At

-SAM, the relaxation constant was found to be smaller by a factor

of 2.5 as compared to the value in H

O. Next, we carried out dis-

tance-dependent TR SERR measurements in H

O buffer solution

including 6.5% sucrose to match the viscosity of D

O. Again, no

changes of the relaxation constants were noted at long chain

lengths whereas at C

-SAM a value of 100 s

1

was determined that

is smaller by a factor of 1.3 than in H

O buffer in the absence of

sucrose. The same ratio of the relaxation constants was found for

-SAM in the presence and in the absence of sucrose.

On the other hand, step-scan SEIRA experiments on C

-SAM-

coated Au electrodes afford a value for k

relax

of 800 s

1

that does

not exhibit H/D or viscosity sensitivity within the experimental

error of 10%.

3.4. Overpotential-dependence of the heterogeneous electron transfer

rate constant

The relaxation constants of the interfacial redox process of

Cyt-c, k

relax

, were determined as a function of the overpotential

for C

-SAM-coated electrodes in H

O buffer. Here, we have

employed potential jumps from an initial potential E

to a

ﬁnal potential E

such that the amount of the overpotential

E

was stepwise increased. Such measurements have been

previously carried out with Cyt-c immobilised on SAM-coated Ag

electrodes (Table 2)[27]. In this work, we have extended the

experiments to C

-SAM-coated Au and Au–SAM–Ag hybrid elec-

trodes, probing the relaxation process by rapid-scan SEIRA and

TR SERR spectroscopy, respectively. SEIRA spectroscopy allows

monitoring the redox process of the immobilised Cyt-c on the basis

of various redox-sensitive amide I bands at 1693 and 1673 cm

1

which have been assigned to the b-turn type III segment 67–70

of the reduced and oxidised Cyt-c, respectively (Figs. 6 and 7)

[13]. For zero-driving force (

= 0 V), k

relax

was determined to be

0.29 s

1

using the average of the relaxation constants derived from

the intensity changes of the individual redox-sensitive amide

bands (Table 2). This value is by a factor of 2 larger than the corre-

sponding value determined for Ag electrodes under otherwise

identical conditions. Increasing the driving force, however, leads

to a much smaller increase of k

relax

as compared to the C

-SAM-

coated Ag electrode with 0.35 and 0.77 s

1

=0.24 V and

=0.44 V, respectively. A further increase of the driving force

(

=0.49 V) causes only a slight increase of k

relax

(Table 2). These

observations are in sharp contrast to the results obtained for the

-SAM-coated Ag electrode for which a substantially larger over-

potential-dependent increase of k

relax

was noted [27].

A comparably unprecedented behaviour is found for the

Au–SAM–Ag hybrid electrode [45,50]. This device is based on an

electrochemically roughened Ag electrode, coated by a SAM of

11-amino-1-undecanethiol (AUT) which in turn is covered by a

15-nm thick Au ﬁlm. The Au surface is then functionalised by a

(carboxyl-terminated) C

-SAM to allow for Cyt-c binding. The

hybrid electrodes offer the advantage of combining the optical

properties of Ag with the electrochemical stability of Au [45].

Moreover, the dielectric spacer between the two metals promotes

plasmon excitation such that the intensity of the SERR spectrum of

Cyt-c immobilised on the outer C

-SAM layer of the Au ﬁlm is

comparable to that observed for Cyt-c at C

-SAM Ag electrodes.

The SERR spectra with 413-nm excitation afford qualitatively the

same results as observed for the Ag-only electrode system, i.e. with

no contribution from non-native species at long chain lengths

[45,50]. ET of the immobilised Cyt-c on C

-SAM-coated

Fig. 5. Relaxation constants for the interfacial redox process of Cyt-c at zero-driving

force at Ag electrodes coated with SAMs of different thickness, expressed by the

number of methylene groups. The solid squares (dark gray), open triangles (blue),

and open circles (red) symbols refer to the data obtained in H

O, H

O/sucrose (1.2

cp), and D

O buffer, respectively. The dotted lines are just to guide the eye. Further

details of the experiments and the data analysis are given in the text. (For

interpretation of the references to colour in this ﬁgure legend, the reader is referred

to the web version of this article.)

Table 2

Relaxation constants for the interfacial redox process as a function of the overpo-

tential of Cyt-c immobilised on different electrode systems.

SAM Ag (SERR)

Au (SEIRA) Au–SAM–Ag (SERR)



(V) k

relax

1

)

(V) k

relax

1

)

(V) k

relax

1

)

0 0.15 0 0.29 0 0.77

0.05 0.26 0.05 1.2

0.1 0.60 0.1 1.2

0.13 0.80

0.2 1.5 0.24 0.35 0.2 4.9

0.3 2.88 0.3 8.2

0.4 3.73 0.44 0.77 0.45 9.0

0.6 3.91 0.49 0.83 0.5 9.6

053 072 045

0.19 240

0.22 320 0.25 180 0.2 167

0.29 370

0.39 500 0.4 313

0.44 550

The data were obtained by TR-SERR experiments with potential jumps from

+0.1 V to different ﬁnal potentials E

corresponding to an overpotential of

E

The average error associated with the determination of k

relax

was ±10%. The k

values for Cyt-c on C

-SAM-coated electrodes as discussed in the text were cal-

culated according to Eq. (2).

Data taken from Refs. [27,42].

H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376 371

Au–SAM–Ag electrodes proceeds via different regimes. For the oxi-

dation reaction, the ﬁrst step is electron tunnelling from Cyt-c

through the outer C

-SAM coating to the Au ﬁlm, followed by

metallic conductance across the Au ﬁlm in the second step, and,

in the third step, again by electron tunnelling through the AUT

SAM to the Ag electrode. The electron transport through the metal-

lic Au ﬁlm is much faster than the tunnelling processes for which

the rates depend on the thickness of the respective SAMs. Thus,

one intuitively expects that the ultimate rate-limiting step is tun-

nelling through the outer C

-SAM coating. This conclusion is in

fact conﬁrmed by a recent systematic analysis of the ET processes

of Cyt-c at C

-SAM-coated Au–SAM–Ag devices in which the

thickness of the inner and the outer SAMs was varied [50]. Conse-

quently, we can compare the ET dynamics of Cyt-c on C

-SAM-

coated Au–SAM–Ag electrodes directly with those at C

-SAM-

coated Au and Ag electrodes. In this respect, the experimentally

determined rate constant is found to be higher by a factor of ca.

2.6 and 5.1 than for the Au and Ag electrode system, respectively.

However, the overpotential is weaker than for Ag albeit more pro-

nounced than for Au (Table 2).

Analogous experiments were carried out with Cyt-c immobi-

lised on C

-SAM coatings for all three electrode systems. The cor-

responding values for k

relax

are listed in Table 2.

4. Discussion

Electrostatic binding of Cyt-c to electrodes coated with car-

boxyl-terminated SAMs proceeds mainly via two binding domains

[46,48]. The high afﬁnity domain (BD3), involving the lysine resi-

dues 72, 73, 79, 86, and 87, is, however, associated with a distinctly

lower average electron tunnelling probability than the medium-

afﬁnity binding domain (BD2), deﬁned by lysine residues 25 and

27. Thus, optimum electron tunnelling requires the rotational dif-

fusion of the immobilised Cyt-c, which at C

-SAM-coated Ag elec-

trodes proceeds with the rate constant k

orient

larger than 6000 s

1

as determined by TR SERR spectroscopy with 514 nm excitation

[42]. This approach is based on the orientation-dependent prefer-

ential enhancement of heme modes of different symmetry under

Q-band excitation. In contrast, the kinetic data obtained by TR

SERR spectroscopy with 413-nm (Soret band) excitation as used

in this work represent relaxation constants k

relax

describing the

reduction and oxidation of the heme. The latter experiments afford

the relaxation of the individual oxidised and reduced Cyt-c species

following a potential jump. This relaxation is generally described

by a mono-exponential function

ðdÞ¼A

ðE

Þexpðk

relax

dÞð1Þ

where A

(d) and A

) are the concentration of the species under con-

sideration at the delay time dand at the initial potential E

(d= 0 s).

For C

-SAM-coated Ag electrodes, k

relax

is 0.15 s

1

at zero-driving

force and it increases to nearly 4 s

1

upon raising the modulus of

the overpotential [27]. Thus, k

orient

is always distinctly larger than

relax

implying that at C

-SAM-coated Ag electrodes the TR-SERR

experiments directly probe electron tunnelling. In a ﬁrst approxima-

tion, this is also true for C

-SAM coatings where, at zero-deriving

force, k

relax

is ca. 50 s

1

compared to k

orient

= 390 s

1

[42]. However,

with increasing driving force, k

relax

approaches k

orient

which be-

comes the limiting value of the overpotential-dependence of k

relax

For C

-SAM coatings, even at zero-driving force, the same values

were determined for k

relax

and k

orient

[42]. Under these conditions,

the overall kinetics of the interfacial ET is the result of the convolu-

tion of protein dynamics and electron tunnelling and should, in fact,

follow a more complex kinetic behaviour that deviates from a mono-

exponential decay [49]. However, within the experimental accuracy

and due to the limited time-resolution of the TR-SERR experiments,

the description of the relaxation processes does not justify ﬁt func-

tions that are more complex than a single exponential.

On the other hand, the increasing deviations for the values of

relax

for C

-SAM coatings with x610 determined in this work

and those obtained earlier in our group (Table 2,[12]) may be re-

lated to the fact that the mono-exponential approximation be-

comes less accurate for thinner SAM coatings. In contrast to the

ﬁt of Eq. (1) to the data in this work, we have previously employed

a ﬁt of the linearized Eq. (1) [12], which might overestimate fast

events in the case of a complex kinetic behaviour. As a conse-

quence, the previous study afforded increasingly larger k

relax

values

for C

-SAM coatings with x610, compared to the present results.

Fig. 6. Rapid scan SEIRA difference spectra of Cyt-c immobilised on a C

-SAM-

coated Au electrode for a potential jump from +0.125 V to 0.4 V. The reference

spectrum refers to the initial potential of +0.125 V. The individual spectra that are

displayed refer to delay times of 0.5, 1, 2.5, 5, and 15 s. The solid arrows indicate the

direction of increasing delay times. Further details of the experiments are given in

the text.

Fig. 7. Time-dependent changes of the intensities of the 1693 and 1673-cm

1

bands determined from rapid-scan SEIRA difference spectroscopy of Cyt-c on C

SAM-coated Au electrode. The data were obtained from potential jump experiments

from +0.125 V (E

)to0.4 V (E

). The reference spectrum refers to the initial

potential of +0.125 V. Further details of the experiments are given in the text.

372 H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376

In addition, the present spectra analysis is not restricted to the

and

band region as previously [12] but covers a wider spectral

range, thereby increasing the accuracy speciﬁcally for thinner

SAMs where contributions from the non-native states gain impor-

tance. This different spectra analysis may also account for the

slight differences of the redox potentials determined from station-

ary SERR experiments (Table 1,[26]).

4.1. Electric-ﬁeld effects on electron tunnelling

At C

-SAM-coated Ag electrodes, i.e. in the tunnelling regime,

relax

determined from the TR-SERR experiments is related to the

electron tunnelling rate constant k

for the reduction of the heme

according to

¼k

relax

K

ð1þK

Þð2Þ

where K

is the redox equilibrium constant

¼exp ðEE

Þne

! ð3Þ

with E,e,k

, and T denoting the electrode potential, the elementary

charge, the Boltzmann constant, and the temperature, respectively.

Thus, for zero-driving force corresponding to

=EE

= 0 V, we

obtain k

=2k

relax

Eqs. (2) and (3) also hold for the relaxation constants derived

from the mono-exponential ﬁts to intensity changes of the SEIRA

bands at 1693 and 1673 cm

1

measured for Cyt-c on C

-SAM-

coated Au electrodes, since these changes seem to proceed instan-

taneously with the heterogeneous ET [13]. This conclusion is very

plausible as the amide I band changes most likely reﬂect the redox-

linked protein structural changes, i.e. the protein reorganisation,

which occurs much faster than long-distance electron tunnelling

considered in this work. Furthermore, even for C

-SAM-coated Au

electrodes, k

relax

at zero-driving force was found to be unaffected

by an increase of the viscosity to 1.2 cp, implying that protein re-

orientation is faster than electron tunnelling. Thus, we can safely

conclude for both the Ag and the Au electrode, the C

-SAM coating

refers to a regime, in which, regardless of the overpotential, elec-

tron tunnelling is the rate-limiting step. The same considerations

hold for the Au–SAM–Ag electrode.

Electron tunnelling between an immobilised redox site and the

electrode can be described according to Eq. (4) [64,65]

Þ

hjVj



expðbdÞerfc kþe

ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4kk

! ð4Þ

where k,V, and

are the reorganisation energy, the electronic

coupling parameter, and the density of states in the electrode, respec-

tively, and erfc(z) is the complementary error function. The exponen-

tial function in Eq. (4) represents the distance-dependence of the

electronic coupling parameter with d and bdenoting the ET distance

and the tunnelling decay parameter. Upon relating the ET rate con-

stant at a given overpotential k

(

) with the rate constant at zero-

driving force (

= 0 V), k

(0), one obtains

ð0Þ¼

erfc

kþe

ﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4kk



erfc

ﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4kk

 ð5Þ

which has been employed to determine the reorganisation energy

for the electron tunnelling process of Cyt-c immobilised on a

-SAM-coated Ag electrode (Fig. 8)[27]. In this way, a reorganisa-

tion of 0.22 eV has been determined.

We now consider the overpotential-dependence of k

deter-

mined for Cyt-c on C

-SAM-coated Au and Au–SAM–Ag hybrid

electrodes, determined by rapid-scan SEIRA and TR SERR spectros-

copy, respectively. In both cases, k

displays a drastically weaker

increase with increasing |

|(Fig. 8). This discrepancy with respect

to Ag can hardly be rationalised on the basis of Eq. (5) since it

would imply a physically meaningless low value for reorganisation

energy, speciﬁcally for the SAM-coated Au electrode. Instead, in a

ﬁrst approximation, one may expect that kshould be similar for

Cyt-c, independent of the kind of the supporting metal (vide infra).

The crucial electrochemical property that is different for SAM-

coated Au and Ag electrodes is the potential of zero-charge E

pzc

This quantity depends of the crystalline structure of the metal

and the type of SAM-coating and has been determined to be –

0.45 and –0.2 V for C

-SAM-coated Ag and Au–SAM–Ag electrodes

[50]. It is reasonable to assume that the values for the respective

-coated electrodes are very similar [66]. Correspondingly, we

assume E

pzc

= –0.05 V for C

-SAM-coated Au electrodes taking into

account literature data [66]. The difference between the actual

electrode potential Eand E

pzc

is proportional to the electric ﬁeld

in the SAM/Cyt-c interface [26,54]. Thus, the ET reactions of Cyt-

c at Au and Ag electrodes take place under the action of opposite

electric ﬁelds, and moreover, experience increasing ﬁeld strengths

for Au but decreasing strengths for Ag upon increasing the driving

force for reduction, i.e. with increasingly negative overpotential

=EE

, taking into account the redox potentials of 0.01 V

and +0.04 V for Cyt-c on C

-SAM-coated Ag and Au electrodes,

respectively [12,13]. Accordingly, the electric ﬁeld variation for

the Au–SAM–Ag electrode lies in between those for the Ag and

Au electrode as it ﬁrst decreases until E=E

= +0.04 V [50])

and then increases again for E<E

pzc

As shown previously for the charge recombination in photosyn-

thetic systems, ET transfer reactions may be accelerated or slowed

down by an externally applied electric ﬁeld (e.g. [67–69]). These

and related experimental data have been attributed to an elec-

tric-ﬁeld dependent variation of the free-energy term of the Mar-

cus equation [67,70], as the electric-ﬁeld dependence of the

electronic parameter is usually very small [71]. Accordingly, we

have adopted these approximations and used a simple electric-

ﬁeld dependent correction to the free-energy term to rationalise

Fig. 8. Overpotential-dependence of the heterogeneous electron transfer rate

constant k

for Cyt-c immobilised on C

-SAM-coated electrodes. The black (open

circles), dark gray (open triangles), and light gray (solid squares) symbols refer to

the data obtained from an Ag (TR SERR), Au–SAM–Ag (TR SERR), and Au electrode

(rapid-scan SEIRA) (Table 2). The solid line represents a ﬁt of Eq. (5) to the

experimental data for the Ag electrode, taken from Ref. [27], whereas in the case of

Au and Au–SAM–Ag, dotted lines are included to guide the eyes. Further details of

the experiments and the data analysis are given in the text.

H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376 373

the present kinetic data. These approximations imply that, due to

the fast protein re-orientation at C

-SAM coatings (vide supra),

any electric-ﬁeld dependent differences in the orientational distri-

bution of the immobilised Cyt-c on the three electrode systems

have no effect on the ET kinetics, i.e. ET always takes place via

the optimum electron coupling.

Assuming a linear relationship between E

pzc

and the electric

ﬁeld strength [26,54], the actual driving force may be given by

an effective overpotential

eff

according to Eq. (6)

eff

þ½a

þa

ðEE

pzc

Þ ¼ ðEE

Þþ½a

þa

ðEE

pzc

Þ ð6Þ

where a

and a

are constants. Then Eq. (5) is modiﬁed to

ðEÞ

ð0Þ¼

erfc

kþeðEE

Þþea

þa

ðEE

pzc

½

ﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4kk



erfc

kþea

þa

ðE

E

pzc

½

ﬃﬃﬃﬃﬃﬃﬃﬃﬃ

4kk

 ð7Þ

Eq. (7) is then ﬁtted to the experimental data for C

-SAM-coated Ag

and Au–SAM–Ag electrodes setting k= 0.22 eV [27] in both cases

and using the experimentally determined redox potentials of

0.01 and +0.04 V for Cyt-c on C

-SAM-coated Ag and Au–SAM–

Ag electrodes, respectively [26,50]. Thus, a good and consistent

description is achieved with the same value for a

(ca. 0.01) and

of ca. 0.004 and 0.04 V for Ag and Au–SAM–Ag, respectively

(Fig. 9). With these parameters, the ratio of the ET rate constants

at zero-driving force for Cyt-c at C

-SAM-coated Ag and Au–

SAM–Ag, k

(0, Ag)/k

(0, AuAg), is calculated to be ca. 0.4 which

is in qualitative agreement with the experimental value of 0.2

(Table 2).

The low number of experimental values for k(

) of Cyt-c at the

-SAM-coated Au electrode does not justify a ﬁt of Eq. (7) with

two adjustable parameter but introducing the additional con-

straint of k

(0, Ag)/k

(0, Au) = 0.5 (Table 2), it turns out that a rea-

sonable reproduction of the experimental data may be achieved by

a similarly low value of a

as for Ag and Au–SAM–Ag but a dis-

tinctly more negative value for a

Despite the crudeness of the present approach, the results allow

concluding that electron tunnelling is controlled by the interfacial

electric ﬁeld. This electric-ﬁeld dependence does not only account

for the different overpotential-dependencies at the various elec-

trodes. In addition, it provides a satisfactory explanation also for

the different k

(0) values although for this quantity, electrode-

speciﬁc differences of

and V cannot be neglected a priori. How-

ever, the differences for the electronic densities of states

of the

three electrode systems are expected to be relatively small

[72,73] and, in particularly, cannot account for variations of

(0) by factor of more than 5. This is also true for the effect of

the different tilt angles of the mercaptanes on Ag and Au surfaces

[74–76], which in principle might alter the electronic coupling V.

However, in particular the 2.5 larger k

(0) for Au–SAM–Ag com-

pared to Au argues against this interpretation. Thus, we conclude

that also differences for k

(0) at the various electrodes are largely

due to the different interfacial electric ﬁelds.

Note that for all three electrodes, the parameter a

was found to

be very small. Since this quantity can be considered a correction

term for the reorganisation energy, it implies that kis largely the

same for all three systems and thus independent of the electric

ﬁeld (vide supra)[70]. A more speciﬁc and quantitative description

of the electric-ﬁeld effect, however, would require a more elabo-

rate description of the interfacial electric ﬁeld which is beyond

the scope of the present work.

Extending this analysis to the overpotential measurements to

-SAM-coated electrodes (Table 2) is problematic due to the

interferences of electron tunnelling with protein dynamics which

for Ag has been shown to proceed on comparable time scales al-

ready at moderate overpotentials [42].

4.2. Protein re-orientation and reorganisation of the hydrogen bond

network in the protein/SAM interface

At C

-SAM coatings at Ag electrodes with x65, the interfacial ET

is controlled by the orientational distribution and dynamics of the

immobilised protein. In fact, the dynamics of the overall redox pro-

cess is viscosity-dependent as reﬂected by the decrease of k

relax

by a

factor of 1.3 from the pure H

O buffer to the sucrose containing solu-

tion at 1.2 cp. This decrease is the same for C

-SAM and C

-SAM coat-

ings at the Ag electrode, implying that a potential electric-ﬁeld

dependence of the viscosity effect is beyond the detection limit.

Conversely, the decrease of k

relax

induced by substituting H

against D

O buffer varies with the thickness of the SAM. Whereas

at C

-SAM coatings on Ag electrodes k

relax

O)/k

relax

O) is 1.3

and thus identical to the viscosity-induced decrease of the relaxa-

tion constant, the ratio increases to 2.5 at C

-SAM coatings. Taking

into account the intrinsically higher viscosity of D

O buffer solu-

tion that corresponds to that of a H

O buffer/sucrose solution at

1.2 cp, one may evaluate a true H/D kinetic isotope effect (KIE) at

-SAM coatings of ca. 2.0. At C

-SAM coatings, however, the ob-

served decrease of k

relax

in D

O appears to be largely a viscosity ef-

fect. An upper limit of the H/D KIE in that case, however, may be

estimated to be 1.1, taking into account the experimental accuracy

of the determination of k

relax

. In this respect, the KIEs determined

in this work are actually somewhat smaller than the previously

reported values for which the viscosity contribution was not

Fig. 9. Potential-dependence of the heterogeneous electron transfer rate constant

for Cyt-c immobilised on C

-SAM-coated Ag (top) and Au–SAM–Ag (bottom)

electrodes. The black (open circles) and dark gray (open triangles) symbols refer to

the data obtained from a Ag and Au–SAM–Ag electrode, respectively. The solid lines

represent ﬁts of Eq. (7) to the experimental data. Further details of the experiments

and the data analysis are given in the text.

374 H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376

considered [12]. For C

-SAM-coated Au electrodes, TR SEIRA exper-

iments reveal no viscosity dependence and no H/D KIE for k

relax

which in addition is distinctly larger (800 s

1

) than for Ag. This

ﬁnding indicates that at C

-SAM-coated Au electrodes protein re-

orientation is much faster than on the Ag electrode and does not

appear to constitute the rate-limiting step of the interfacial redox

process.

An increased viscosity is likely to slow-down the large-scale

motions of the immobilised protein, speciﬁcally the change of

the binding site via rotational diffusion [42,46]. However, also

small-amplitude ﬂuctuations of water molecules in the Cyt-c/

SAM contact region are affected as concluded from recent studies

on covalently attached Cyt-c that as well reveals a viscosity effect

of k

relax

in the non-tunnelling regime [47].

The molecular origin of the H/D KIE may be based on two differ-

ent processes. First, the above-mentioned ﬂuctuations of water

molecules in the interface between Cyt-c and the SAM have been

shown to be important for a ﬁne adjustment of the protein

orientation with respect to the surface and thus for a more efﬁcient

electronic coupling [46,48]. Such motions inevitably include a

re-arrangement of the hydrogen bond network, which is part of

the thermal ﬂuctuations of the system and is not a pre-requisite

for the ET although they may affect the rate of electron tunnelling.

Second, the change of the charge distribution at the redox site

associated with the ET requires a re-arrangement of the hydrogen

bond network, speciﬁcally albeit not exclusively, in the immediate

surroundings of the heme as a part of the structural reorganisation

[12,27]. It might be that this process involves short-range proton

translocations, corresponding to a proton-coupled electron trans-

fer mechanism. Such a mechanism has been proposed in our previ-

ous study [12,27] and, recently, discussed in more detail by Davis

and Waldeck [43] in the context of further experimental and theo-

retical data [30,77,78]. In any case, the fact that the same KIE is ob-

served regardless of the H/D exchange method (vide supra) implies

that the kinetically relevant protons are easily exchanged.

Both processes, the ﬂuctuations of the hydrogen bond network

in the Cyt-c/SAM interface, and the redox-linked re-arrangement of

the internal hydrogen bond network, may account for the H/D KIE

effects observed in this work. In each case, displacements of

(partially) charged groups and protons are involved which are

likely to be affected by the interfacial electric ﬁeld. The decrease

of the H/D-sensitive relaxation constant from C

-SAMs to C

-SAMs

points to an electric-ﬁeld dependent increase of the activation

energy required for the underlying processes. Moreover, also the

KIE itself increases with increasing ﬁeld strength (i.e., from C

-to

-SAMs).

4.3. Overall electric-ﬁeld effect on the interfacial electron transfer

process

Due to the electric-ﬁeld dependent decrease of the protein re-

orientation rate, the experimentally determined values for k

orient

and k

relax

are essentially the same for C

-SAM-coated Ag electrodes

[42]. Under these conditions, the interfacial redox process may be

described by a convolution of protein re-orientation and orienta-

tion-dependent electron tunnelling probabilities [42,48]. Most

likely, the majority of the proteins is bound to the SAM surface

via the high afﬁnity domain BD3 for which the average electronic

coupling parameter is predicted to be lower by a factor of ca.10

as compared to the medium-afﬁnity binding domain BD2 for

which the largest average electron coupling parameter has been

calculated [46]. As a consequence, electron tunnelling should be

faster by a factor of 100 for Cyt-c bound via BD2 than via BD3.

Thus, the lower limit for the heterogeneous ET rate (slow protein

re-orientation) should be given by the average electron tunnelling

rates via BD3 whereas the upper limit (fast protein re-orientation)

results from electron tunnelling via BD2. Then, the upper limit of

the ET rate at C

-SAM coatings may be estimated on the basis of

distance-dependent term in Eq. (4) by extrapolating the rate con-

stants in the electron tunnelling regime (C

- and C

-SAMs) to

the C

-SAM system [12]. This leads to a limiting rate constant of

610

1

that is four orders of magnitude larger than the exper-

imentally observed value for k

relax

. This difference has to be com-

pared with the gap of two orders of magnitude between the

lower and upper limit for orientation-dependent tunnelling as de-

rived from the MD simulations. Moreover, this discrepancy is even

more severe considering the kinetics at the C

-SAM since the

experimental value for k

relax

even drops compared to the C

-SAM.

Just due to the distance dependence one would have expected an

increase of the rate constant by a factor ca. 500, independent of

the protein orientation and orientational dynamics.

These considerations imply that with increasing electric ﬁeld

strength at short distances the electron tunnelling rate is drasti-

cally slowed down. Most likely, in this high-ﬁeld regime, the elec-

tric-ﬁeld dependence of the tunnelling rate is more complex than

included in Eqs. (7) and (6). Speciﬁcally, the assumption of an elec-

tric-ﬁeld independent electronic coupling parameter (Eq. (4))

might not be justiﬁed. In fact, the effect of the interfacial electric

ﬁeld on the electronic coupling parameter has not been considered

in the previous calculations of the electron tunnelling probabilities

for the various orientations of Cyt-c [46,48].

On the basis of this interpretation one can also understand the

ca. 1000 times smaller relaxation constant for SAMs of the same

length as C

but which carry a phosphonate instead of a carboxyl-

ate head group [15]. Due to the signiﬁcantly higher charge density

on the phosphonate-terminated SAM, the interfacial electric ﬁeld

strength is larger than for the carboxyl-terminated SAM, causing

the drastic decrease of the electron tunnelling rate constant. The

same explanation holds for the ca. 40 times lower k

at Ag elec-

trodes covered with sulphate anions compared to the C

-coated

Ag electrode [58].

The interfacial redox process of Cyt-c on electrodes involves

four different elementary steps, that are electron tunnelling cou-

pled with the reorganisation of the protein structure, protein re-

orientation on the SAM surface, and the re-arrangement of the

hydrogen bond network in the protein/SAM interface and possibly

also inside the protein. The individual steps depend on the local

electric ﬁeld, which in turn is controlled by the distance- and po-

tential-dependence of the surface charge density on the SAM

and of the quantity (EE

pzc

)[26,54]. The interplay of these elec-

tric-ﬁeld dependent steps results in a complex overall potential-

and distance-dependent kinetic behaviour.

Since the surface change density

and (EE

pzc

) are different

for different metals (i.e. Ag and Au) and the different types of coat-

ings (i.e., charged, polar, or hydrophobic SAM surfaces) one may

readily understand why the experimentally determined rate con-

stants may differ among the various electrode/SAM systems but

display, in toto, a similarly shaped distance-dependence [12,15,

23,25,29–32,36].AtC

-SAM-coated Au electrodes, for instance,

relax

at zero-driving force (E=E

) is more than 6 times larger than

in the case of Ag. This difference can be attributed to the fact that

E

pzc

| is smaller for Au and, hence, also the electric ﬁeld is

weaker for Au than for Ag (vide supra) such that both electronic

tunnelling and, as supported by the lack of a signiﬁcant viscosity

and H/D KIE effect, also protein re-orientation and hydrogen bond

ﬂuctuations are faster. On the other hand, an electrochemical

investigation by Davis and Waldeck [43] reported a viscosity-

corrected H/D KIE of ca. 1.2 for Cyt-c immobilised on Au electrodes

coated by mixed carboxyl- and hydroxyl-terminated SAMs of 5 and

4 methylene groups, respectively. This difference compared to the

present results is evidently due to the mixed monolayer that,

compared to the pure carboxyl-terminated SAM used in this work,

H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376 375

affords a ca. 6 times larger k

relax

, close to the rate constants of the

viscosity- and H/D-dependent processes.

5. Conclusions

We have shown that the electric-ﬁeld effects on protein motion,

electron tunnelling, and hydrogen bond re-arrangements provide

the basis for a consistent description of the present and previous

experimental ﬁndings on the heterogeneous ET reaction of Cyt-c

obtained for different electrode systems and SAM-coatings

[12,15,23,25,29–32,36]. Moreover, these electric-ﬁeld effects seem

to constitute a general mechanism for interfacial processes of quite

different redox proteins, since in each case the same elementary

steps are involved. Details may be different depending on the sur-

face charge of the protein’s binding domains that may be either

cationic, anionic, or hydrophobic and may contain heme or copper

redox centers [51–55].

Acknowledgement

The work was supported by the DFG (Cluster of Excellence –

UniCat).

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376 H. Khoa Ly et al. / Journal of Electroanalytical Chemistry 660 (2011) 367–376

Perturbation of the Redox Site Structure of Cytochrome c

upon Tyrosine Nitration

Ly, H. K.; Utesch, T.; Díaz-Moreno, I.; Garía-Heredia, J. M.; Rósa, M. A. d. l.;

Hildebrandt, P. Pertubation of the Redox Site Structure of Cytochrome cupon Tyrosine

Nitration. J. Phys. Chem. C 2012, 116, 5694–5702.

Perturbation of the Redox Site Structure of Cytochrome cVariants

upon Tyrosine Nitration

H. Khoa Ly,

†

Tillmann Utesch,

†

Irene Díaz-Moreno,

‡

JoseM. García-Heredia,

‡

Miguel Angel De La Rosa,

‡

and Peter Hildebrandt*

,†

†

Institut fur Chemie, Technische Universitat Berlin, Sekr. PC14, Straße des 17 Juni 135, D-10623 Berlin, Germany

‡

Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevialla-CSIC, Avda Americo Vespucio 49, Sevilla 41092,

Spain

SSupporting Information

ABSTRACT: Post-translational nitration of tyrosine is

considered to be an important step in controlling the multiple

functions of cytochrome c(Cyt-c). However, the underlying

structural basis and mechanism are not yet understood. In this

work, human Cyt-cvariants in which all but one tyrosine has

been substituted by phenylalanine have been studied by

resonance Raman and electrochemical methods to probe the

consequences of tyrosine nitration on the heme pocket

structure and the redox potential. The mutagenic modiﬁca-

tions of the protein cause only subtle conformational changes

of the protein and small negative shifts of the redox potentials

which can be rationalized in terms of long-range electrostatic eﬀects on the heme. The data indicate that the contributions of the

individual tyrosines for maintaining the relatively high redox potential of Cyt-care additive. Nitration of individual tyrosines leads

to a destabilization of the axial Fe−Met80 bond which causes the substitution of the native Met ligand by a water molecule or a

lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-

assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and

non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the

Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of

the heme pocket of Cyt-cdue to tyrosine nitration and to local electric ﬁelds are independent of each other and occur via

diﬀerent molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic

binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the

peroxidase function.

■INTRODUCTION

Post-translational modiﬁcations of tyrosine residues in proteins

represent essential steps for controlling biological functions,

frequently related to signaling pathways of cells. Phosphor-

ylation is known to play a key role in a variety of regulatory

processes in cell growth, diﬀerentiation, and metabolism.

1,2

Nitration of tyrosines has been shown to occur in response to

oxidative stress, induced by reaction with NO species.

2−4

The

heme protein cytochrome c(Cyt-c) is one of the target proteins

for which both types of tyrosine modiﬁcations have been

demonstrated and the eﬀects on Cyt-c's biological functions

have been widely studied.

5−19

Cyt-cserves as an electron carrier in the mitochondrial

intramembrane space transferring electrons from the mem-

brane-bound cytochrome creductase to cytochrome coxidase

where molecular oxygen is reduced to water.

In addition, Cyt-

chas been shown to play crucial roles in programmed cell

death. In one case, Cyt-cbinds to the Apaf-1 protein, thereby

causing its oligomerization and the subsequent activation of

caspases.

Prior to this reaction sequence, however, Cyt-chas

to be transferred through the mitochondrial membrane to the

cytosol, a process that is most likely related to the function of

Cyt-cas a catalyst of H2O2-dependent peroxidation of the

mitochondria-speciﬁc cardiolipin.

22−24

Nitration of tyrosine residues in Cyt-chas been proposed to

be the initial event that triggers the change from the biological

function in the respiratory chain to apoptosis.

6,14

In fact, in

vitro studies revealed an enhanced peroxidase activity for Cyt-c

upon Tyr nitration,

7,8,13

suggesting a transition from the

electron-transfer to cardiolipin-peroxidation function. On the

other hand, Tyr nitration inhibits caspase-9 activation and thus

blocks the apoptosis signaling pathway.

9,13,15

In this respect,

nitration may exert a similar inhibitory eﬀect on caspase as

phosphorylation as judged from studies on phosphomimetic

Tyr mutants.

18,19

Received: March 9, 2012

Revised: April 26, 2012

Published: April 27, 2012

Article

pubs.acs.org/JPCB

Peroxidase activity requires the accessibility of the heme iron

by peroxide which is prohibited by the “native”closed heme

pocket structure of Cyt-cwith the axial coordination sites

occupied by His18 and Met80. It is thus tempting to relate the

catalytically active state of Cyt-cto a ﬁve-coordinated species

lacking the Met80 ligand as it is formed upon electrostatic

binding to model membranes.

25−27

Thus, strong electrostatic

interactions, possibly in concert with Tyr nitration, have also

been suggested to play a key role for the transformation of Cyt-

cfrom an electron carrier to a catalyst.

Clearly, a deeper understanding of the functional switch of

Cyt-cas well as the molecular processes of the protein in

apoptosis requires a profound knowledge of the structural

consequences of Tyr nitration in Cyt-c. Following a previously

established approach,

11,13−15

mutants of human Cyt-chave

been expressed in which four out of ﬁve Tyr were substituted

by Phe, allowing for a selective nitration of the remaining Tyr

(Figure 1). In this work, we have employed resonance Raman

(RR) spectroscopy to analyze the eﬀect of the mutagenic and

chemical modiﬁcations on the redox center, speciﬁcally the

nitration-induced changes in the coordination pattern of the

heme. Furthermore, the protein variants were electrostatically

immobilized on electrodes coated by self-assembled mono-

layers (SAMs) of amphiphiles to mimic interactions with

negatively charged membranes. Using surface-enhanced reso-

nance Raman (SERR) spectroscopy and cyclic voltammetry

(CV), these studies aim at investigating the combined eﬀect of

Tyr nitration and electrostatic ﬁelds on the heme structure as

well as on the redox properties.

■MATERIALS AND METHODS

Materials. 11-Mercaptoundecanoic acid (MUA) and

sodium peroxodisulfate were purchased from Sigma Aldrich.

Potassium hydrogen phosphate, potassium dihydrogen phos-

phate, potassium hydroxide and sodium dithionite were

provided by Fluka (Merck, Germany). Ethanol (99.9%) was

obtained from Fischer Scientiﬁc Company (Germany). Buﬀer

solutions were prepared using Millipore water (Eschborn,

Germany) with a resistance >18 MΩ. All chemicals were used

as received.

Expression of Cyt-cMutants. Recombinant human Cyt-c,

either the wild-type species or the monotyrosine mutants in

which all tyrosine residues but one (just that at the indicated

numbered position, Figure 1) were substituted by phenyl-

alanines, were expressed in E. coli DH5αand further puriﬁed by

ionic exchange chromatography, as previously described.

11,13

Nitration. Peroxynitrite synthesis and nitration of the

diﬀerent Cyt-csamples were carried out as reported

previously,

11,13,14

with the following modiﬁcations: Fe3+-

EDTA concentration and the number of peroxynitrite additions

were increased up to 1.5 mM and 10 bolus additions,

respectively. The nitration reaction was performed under acidic

conditions (pH 5.0). The resulting nitrated Cyt-cspecies were

intensively washed with 10 mM potassium phosphate (pH 6.0).

Nitrated monotyrosine Cyt-cmutants were separated from

non-nitrated protein in a CM-cellulose column equilibrated

with 1.5 mM borate, pH 9.0, using a 0−100 mM NaCl gradient.

Nitrated Cyt-celuted at a much lower salt concentration than

native protein because of the extra negative charge of

deprotonated tyrosyl anions, whose pKais modiﬁed by the

strong electron-withdrawing eﬀect of the NO2substituent at

the 3-position.

The purity to 95% homogeneity of nitrated

Cyt-cpreparations was corroborated by SDS-Page and Western

Blot using antibodies antinitrotyrosine (Biotem) to detect the

presence of the NO2group. In addition, the molecular mass

and the speciﬁcally nitrated tyrosine residue of each mutant

were conﬁrmed by tryptic digestion and MALDI-TOF (Bruker-

Daltonics, Germany) analyses. Samples were concentrated up

to 0.2−2.0 mM in 5 mM sodium phosphate buﬀer (pH 6.0).

Resonance Raman and Surface-Enhanced Resonance

Raman Spectroscopy. RR and SERR spectra were acquired

using the 413 nm line of a krypton ion laser (Coherent Innova

300c) coupled to a single-stage spectrograph (Jobin Yvon,

LabRam 800 HR) equipped with a liquid-nitrogen-cooled back-

illuminated CCD detector. The laser beam was focused using a

Nikon 20×objective (N.A. 0.35) with a working distance of 20

mm. The laser power on the sample was 1 mW. Acquisition

times ranged from 5 to 30 s depending on spectral quality and

sample concentration.

RR measurements were carried out in a rotating quartz

cuvette. Protein solutions were oxidized and reduced chemically

using sodium peroxodisulfate and sodium dithionite, respec-

tively. SERR measurements were conducted in a spectroelec-

trochemical cell described previously.

An electrochemically

roughened silver ring (99%, Gotz, Germany) coated with a

SAM of MUA served as a working electrode. SER activation,

SAM coating of the electrode, and subsequent protein

immobilization followed the protocol described previously

with minor modiﬁcations.

28,29

Brieﬂy, prior to experiments the

electrolyte solution (50 mM phosphate buﬀer at pH = 7.0, 9.0,

or 12.0) was purged with catalytically puriﬁed oxygen-free

argon for ca. 20 min. Then, the protein solution was added,

yielding a ﬁnal concentration of ca. 0.2 μM. Protein adsorption

was achieved by incubating the working electrode for 30 min

into the protein containing buﬀer solution at open circuit. All

SERR experiments were performed in the presence of protein

in solution and under Ar overpressure. Potentials cited in this

Figure 1. (Top) Three-dimensional structure of human Cyt-c(WT)

highlighting the heme and the tyrosine residues 46 (violet), 48

(orange), 67 (green), 74 (red),and97(yellow).(Bottom)

Coordination patterns of the various states of Cyt-c.

The Journal of Physical Chemistry B Article

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worked refer to an Ag/AgCl (3 M KCl) reference electrode

(+0.21 V vs NHE).

Electrochemistry. CV experiments were performed in the

spectroelectrochemical SERR cell (vide supra) connected to a

CH Instrument 660 C potentiostat (Austin, TX). Determi-

nation of peak maxima and baseline subtraction were executed

using the CHI software (CHI Electrochemical workstation,

Version 6.13).

Molecular Dynamics Simulation. The crystallographic

structure of human Cyt-c(3NWV)

served as template for the

molecular dynamics (MD) simulations. The desired mutations

and chemical modiﬁcations (nitration) were incorporated with

VMD 1.8.7.

In addition to these sequence changes, each

mutant carried a glycine instead of a serine at position 41,

which is a naturally occurring apoptosis enhancing mutant.

Details of the MD simulations are given in the Supporting

Information.

■RESULTS

Resonance Raman Spectroscopy. RR spectroscopy is an

ideal tool to probe the consequences of protein modiﬁcations

on the redox center of Cyt-c. The present study is restricted to

the ferric form of Cyt-c, generated by the addition of sodium

peroxodisulfate, although in some cases a complete oxidation

was not achieved. However, remaining traces of ferrous Cyt-c

could readily be identiﬁed in the experimental spectra on the

basis of its strongest band at ca. 1360 cm−1. Besides this minor

interference by the ferrous form, the experimental spectra

particularly of the Tyr-nitrated protein variants display a

substantial heterogeneity as reﬂected by asymmetric band

shapes, shoulders, and extra bands not present in the RR

spectrum of the native Cyt-c(Figure 2).

These spectral changes speciﬁcally in the region of the modes

ν4,ν3, and ν2indicate contributions from spin and coordination

states that diﬀer from the His-Met-coordinated low-spin

conﬁguration of the native protein, denoted as state B1.

For instance, the broad 1477 and 1569 cm−1bands point to six-

and ﬁve-coordinated high-spin (6cHS, 5cHS) species and the

broadening of the bands at 1502 and 1584 cm−1may originate

from non-native six-coordinated low-spin (6cLS) conﬁgura-

tions.

In a ﬁrst attempt to analyze these spectral changes

quantitatively, we assumed that mutations and the respective

tyrosine nitration solely aﬀect the distribution among diﬀerent

spin and coordination states of the heme whereas the spectra of

the individual components are the same in the various protein

variants, i.e., the native 6cLS state B1 and the non-native 6cLS,

6cHS, and 5cHS species.

However, this component analysis

failed to provide a satisfactory global ﬁt to all experimental RR

spectra (see Supporting Information). Each of the amino acid

substitutions and the respective chemical modiﬁcation of the

single tyrosines cause subtle spectral alterations with frequency

changes of ca. ±2cm

−1and intensity variations of ca. 20% for

nearly each of the component spectra. Such variations cannot

be neglected within a global ﬁt, although they are distinctly

smaller than the diﬀerences between the component spectra of

the individual spin and coordination states.

Thus, we have restricted the RR spectra analysis to the ν3

band, which is most sensitive to changes of the spin and

coordination state of the heme iron, and hence allows for a

quantitative analysis in terms of the various coordination states

involved. On the basis of a large body of experimental data, the

frequency ranges of this band of 1478−1481, 1488−1491,

1500−1502, and 1504−1506 cm−1in the ferric state are

indicative of a 6cHS, 5cHS, a Met-His 6cLS, and a “N”-His (N

= His, Lys) 6cLS conﬁguration, respectively.

26,32

We thus have

employed a band-ﬁtting analysis to this spectral region using

four bands with frequencies that were allowed to vary in the

above-mentioned ranges in order to take into account the

speciﬁceﬀects of the individual mutations and nitration (vide

supra) (Figure 3; further details of the band ﬁtting are given in

the Supporting Information). This procedure is associated with

a relatively large error for determining weak contributions of

the 6cHS state in the presence of a prevailing B1 contribution

since the latter species gives rise to a broad band at 1465 cm−1

(ν28) that overlaps with the ν3mode of the 6cHS state. After

taking into account this interference, the relative intensities

obtained by the ﬁtting procedure were then converted into

relative concentrations (Supporting Information)

as listed in

Table 1.

The data for the non-nitrated protein variants indicate no

changes of the coordination and spin state when all ﬁve Tyr

(null mutant) are substituted by Phe. This is also true for the

monotyrosine variants Y46 and Y74. However, a small but

clearly detectable decrease of the native state B1 by ca. 15% is

Figure 2. RR spectra of WT Cyt-c(bottom) and the Y48-NO2variant

(top) in the marker band region.

Figure 3. RR spectra in the ν3band region of the WT Cyt-c(bottom)

and the Y46-NO2variant (top), including the ﬁtted Lorentzian band

shapes.

The Journal of Physical Chemistry B Article

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noted for Y67 and Y48 and by 8% for Y97. The situation is

diﬀerent when the single Tyr residues are nitrated. These

modiﬁcations lead to a signiﬁcant population of a non-native

6cLS and a HS conﬁguration (predominantly 6cHS) with a

relative contribution of ca. 15% and between 25 and 45%,

respectively. The only exception refers to Y97-NO2, in which

the nitration site (i.e., the tyrosine) is most remote from the

heme (Supporting Information). Here the relative contribu-

tions of the non-native species are distinctly lower and are,

within the ﬁtting accuracy, unchanged compared to the non-

nitrated protein.

The non-native 6cLS state is of particular interest since the

nature of the “new”axial ligand that has replaced Met80 is not

clear a priori. The position of the marker bands ν3(vide supra)

or ν10 indicate the coordination via a nitrogen atom that may

either be provided by a His or by a Lys. In the former case,

His33 or His26 may be potential candidates since these amino

acids have been identiﬁed as axial ligands in an (metastable)

intermediate formed during unfolding or refolding.

This

coordination pattern is also found in Cyt-celectrostatically

bound to negatively charged surfaces, denoted as state B2 in

distinction to state B1 as the native state.

Alternatively, a Lys

(72, 73, or 79) may bind to the heme iron corresponding to the

“alkaline”state typically formed for WT Cyt-cat a pH above

10.

35,36

We have, therefore, examined the low-frequency region of

the RR spectra to distinguish between both possibilities. In this

region, the alkaline state and state B2 each exhibits a unique

band pattern that diﬀers from the characteristic RR spectrum of

the native Met-His coordinated 6cLS state (B1) and are not

superimposed by the intrinsically weak RR bands of the HS

species.

35,37

Nevertheless, the relatively low relative contribu-

tions of the non-native 6cLS form require a diﬀerence

procedure to enhance the spectral changes with respect to

the native state.

First, the WT protein was measured at pH 7 and pH 12 to

obtain the native B1 state and a largely pure alkaline state. The

diﬀerence spectrum “pH12 minus pH7”in this region displays a

characteristic pattern of positive and negative signals (Figure 4),

which is also found for the null mutant and all non-nitrated

Cyt-cvariants, indicating that in each mutant the alkaline form

with its characteristic Lys/His axial coordination pattern of the

heme prevails at pH 12. To ﬁnd out whether the non-native

6cLS state, which is partially populated upon Tyr nitration in

neutral solutions, exhibits the same Lys/His coordination as the

alkaline form, we have constructed the diﬀerence spectra

“nitrated monotyrosine mutant minus non-nitrated monotyr-

osine mutant”from the spectra measured at pH 7. In fact, the

diﬀerence spectra for all tyrosine mutants (i.e., “nitrated”minus

“non-nitrated”) display band patterns, which are similar to that

of the “pH12 minus pH7”diﬀerence spectrum of WT Cyt-cor

the “null”mutant (Figure 5). These ﬁndings suggest that in the

nitrated variants the N-containing ligand of the “N”/His 6cLS

species is a Lys.

UV−Vis Absorption Spectroscopy. The eﬀect of the Tyr-

to-Phe substitutions on the absorption spectra of the non-

nitrated Cyt-cvariants in the Soret band region is relatively

small (ca. <3 nm), whereas upon nitration the peak maximum

is shifted more signiﬁcantly to the blue by up to 6 nm

(Supporting Information). These shifts mainly reﬂect the

coexistence of diﬀerent spin and coordination states and

speciﬁcally the formation of a HS species which is known to

exhibit a Soret band maximum at wavelengths shorter than the

6cLS forms. Thus, the UV−vis absorption data are in

agreement with the RR spectroscopic results inasmuch as the

largest blue shift of the Soret maximum is noted for Y46-NO2

and Y48-NO2which includes the largest HS contribution, in

agreement with previous reports.

14,15

Surface-Enhanced Resonance Raman Spectroscopy.

To probe electric ﬁeld eﬀects on the protein stability, Cyt-c

variants were electrostatically immobilized on an Ag electrode

coated with a carboxyl-terminated SAM of MUA and studied by

SERR spectroscopy. The SERR spectrum of WT Cyt-cis

Table 1. Relative Contributions of the Various Spin and

Coordination States in Nitrated and Non-nitrated Cyt-c

Variants (in %)

native non-native

B1 6cLS

(Met-His) 6cLS

(Lys-His) HS

nitration-induced destabilization

of the native state B1

WT 100 0 0 −

null 100 0 0 −

Y46 100 0 0 −

Y46-

NO2

42 42 2.4

Y48 85 4 11 −

Y48-

NO2

40 13 47 2.1

Y67 86 0 14 −

Y67-

NO2

57 12 31 1.5

Y74 100 0 0 −

Y74-

NO2

63 13 24 1.6

Y97 92 1 7 −

Y97-

NO2

90 4 6 1.0

The contribution of the 5cHS (-His) and 6cHS (H2O-His) were

combined.

Ratio of the contributions of the native state in the non-

nitrated and nitrated forms

Figure 4. Low-frequency RR spectra of WT Cyt-cat pH 12 and at pH

7. The third trace displays the diﬀerence spectrum “pH 12 minus pH

7”.

The Journal of Physical Chemistry B Article

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dominated by the component spectrum of the native state B1

which is nearly identical to the RR spectrum of the protein in

solution. This state could be reversibly oxidized and reduced by

varying the electrode potential. Thus, the behavior of the

human WT Cyt-con MUA-coated electrodes is similar to that

of horse heart protein except for a higher contribution of non-

native states in the SERR spectrum of human Cyt-c.

Unlike the RR spectroscopic analysis of the proteins in

solution, a global component analysis could be applied starting

with the component spectra previously determined for horse

heart Cyt-c.

26,28

Only minor adjustments of a few spectral

parameters (i.e., intensities in the ν2region) were required to

improve the global ﬁt. Thus, all experimental spectra could be

simulated by the same set of component spectra, i.e., the

spectra of the native reduced and oxidized B1 state (6cLS with

Met-His ligation) and the oxidized B2 species including the

5cHS (−/His) and 6cHS (H2O/His) conﬁguration as well as

the 6cLS (“N”/His) conﬁguration (see Supporting Informa-

tion). The component spectrum of the latter is nearly identical

to that of the bis(histidine)-coordinated 6cLS state of horse

heart Cyt-cimplying that, unlike the proteins in solution, an

additional His (instead of a Lys) serves as the sixth ligand in the

immobilized nitrated and non-nitrated Tyr variants. Spectra of

the reduced forms of the B2 species were not required for the

global ﬁt since the experiments were carried out above the

respective reduction potentials between −0.25 and −0.4 V (vs

AgCl).

Figure 6 shows an example of a SERR spectrum

analyzed in this way. The resultant relative weights of the

individual component spectra were then converted into relative

concentrations (Table 2) according to the procedure described

previously (Supporting Information)

such that the data can

be directly compared with those obtained for Cyt-cin solution

on the basis of the ν3band ﬁtting (Table 1). However, the

accuracy of the component analysis is signiﬁcantly higher than

for a simple band ﬁtting analysis since complete spectra of the

individual components are ﬁtted to the experimental spectra

and the degrees of freedom in the ﬁtting procedure are

restricted to the number of components.

Further data on the

distribution among the various states at open circuit of the

electrode are given in the Supporting Information.

Electrochemistry. The formal midpoint potential and the

electron-transfer rate constants of the various Cyt-cvariants

immobilized on SAM-coated Ag electrodes were measured by

CV using a potential range from +0.15 to −0.2 V. Under these

conditions, CV solely probes the redox potentials of the native

B1 state since, according to the previous ﬁndings for horse

heart Cyt-c, the B2 species should give rise to redox potentials

lower than −0.2 V.

Extending this conclusion from horse

heart to human Cyt-cis justiﬁed in view of the far-reaching

similarities in the three-dimensional structures

30,40

and the

spectral properties, including the RR spectra (vide supra). Also,

the midpoint potentials of the immobilized WT proteins have

been found to be essentially the same with 22 and 23 mV for

horse heart and human Cyt-c, respectively.

28,41

Figure 5. Diﬀerence spectrum “pH 12 minus pH 7”of the null mutant

compared with the diﬀerence spectra of Y48-NO2and Y67-NO2

obtained from the spectra of the respective nitrated mutants and the

non-nitrated mutant, all measured at pH 7.

Figure 6. SERR spectrum of the null variant of Cyt-cobtained from a

MUA-coated Ag electrode, including the component spectra obtained

by a global ﬁt. The component spectra of the oxidized B1, reduced B1,

the oxidized B2[6cLS], and the oxidized B2[HS] (sum of the 5cHS

and 6cHS species) are given by the dotted lines in green, magenta,

blue, and red, respectively. The dotted gray line represents the sum of

the component spectra.

Table 2. Relative Contributions (in %) of the Various Spin

and Coordination States of Cyt-cVariants Immobilized on

SAM-Coated Electrodes at +0.15 V

B1 6cLS

(Met-His) B2 6cLS

(His-His) B2HS

electric-ﬁeld-induced

destabilization of the native state

WT 76 16 8 1.3

null 49 39 12 2.0

Y46 42 17 41 2.3

Y46-

NO2

30 24 46 1.4

Y48 42 42 16 2.0

Y48-

NO2

43 43 14 0.9

Y67 51 41 8 1.7

Y67-

NO2

42 43 15 1.4

Y74 64 20 16 1.6

Y74-

NO2

45 42 13 1.4

Y97 42 26 32 2.2

Y97-

NO2

33 21 46 2.7

The contributions of the 5cHS (-His) and 6cHS (H2O-His) were

combined.

Ratio of the contributions of the native state in solution

(Table 1) and in the immobilized state.

The Journal of Physical Chemistry B Article

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The CVs aﬀorded well-resolved peaks and a quasi-reversible

electron transfer (ET), reﬂected by a full width at half-height of

80−100 mV (Figure 7). Upon mutation the redox potential is

signiﬁcantly lowered by up to 50 mV (Table 3). Additional

downshifts of the redox potential were noted upon tyrosine

nitration except for the Y74 protein which displays a small

upshift in the Y74-NO2variant.

Upon varying the scan rate, the ET kinetics of the

immobilized Cyt-cvariants has been determined by applying

the Laviron method (Figure 7). The ET rate constant of the

WT protein was found to be distinctly smaller compared to that

determined for horse heart Cyt-cunder otherwise the same

conditions (4 vs 40 s−1).

The mutation of the individual

tyrosines leads to a strong increase of the ET rate constant

whereas nitration either accelerates or slows down the

heterogeneous ET.

■DISCUSSION

Eﬀect of Phe-to-Tyr Mutations on the Redox Center.

The most striking result of the RR spectroscopic analysis of the

non-nitrated Cyt-cis the fact that substitutions of all Tyr by Phe

leaves the redox site structure essentially unchanged whereas

preservation of a single Tyr residue in position 48, 67, or 97

causes a weakening of the Fe−Met bond and thus leads to small

contributions of non-native states. These results are diﬃcult to

rationalize since one intuitively expects that speciﬁcally Tyr67,

due to its proximity to the heme, exerts a stabilizing function on

the heme pocket. We thus conclude that the small eﬀects on

the coordination state of Cyt-c(as well as on the small spectral

modiﬁcations of the respective native B1 state) are largely due

to long-range perturbations of the electrostatics in the heme

pocket. In some cases, such as the null mutant, the structural

consequences of individual Tyr-to-Phe substitutions may

accidentally compensate each other. However, it should be

noted that this conclusion solely refers to the structure of the

heme site as the RR spectra do not provide information about

structural changes of protein.

The situation is diﬀerent for the eﬀect of the individual

tyrosine residues on the redox potential of the heme in the

native state B1. All the monotyrosine mutants, including the

null variant, display negative shifts of the redox potential with

respect to the WT protein (Table 3). Assuming that each Tyr

residue exerts an incremental contribution to the redox

potential, one may express the redox potential shift ΔEifor

each variant iaccording to

∑ε

=− =EE E

(1)

where EWT

0and Ei

0are the redox potentials of the WT protein

and mono-Tyr variant i, respectively, and εi,jrepresents the

incremental redox potential shift due to the Tyr-to-Phe

substitution at the amino acid position j; i.e., εi,jis zero for i

=jbut nonzero for i≠j. Correspondingly, one obtains a set of

ﬁve equations according to the scheme in Table 4 that can be

solved to aﬀord the individual values for εj. This approach is

justiﬁed a posteriori since the sum of all εjvalues is very similar

Figure 7. CVs of WT human Cyt-c(top) obtained with increasing

scan rates (in the direction of the arrow, 100, 200, 300, 400, 500, 750,

and 1000 mV/s) and the Laviron plot (bottom).

Table 3. Redox Properties of the Non-nitrated and Nitrated

Cyt-cVariants Immobilized on a MUA-Coated Ag Electrode

protein E0/mV kET/s−1

WT 22 4

null −33 29

Y46 −24 59

Y46-NO2−46 31

Y48 −13 10

Y48-NO2−61 28

Y67 −27

Y67-NO2−527

Y74 −28 60

Y74-NO2−20 40

Y97 −22 32

Y97-NO2−31 53

Table 4. Shifts of the Redox Potential of Non-nitrated

Protein Variants and Contributions of the Individual Tyr

Residues

tyrosine residues

protein 67 48 46 74 97 ΔE0/mV

WTxxxxx 0

Y67 x −−−− −24

Y48 −x−−− −35

Y46 −−x−− −46

Y74 −−−x−−50

Y97 −−−−x−44

null −−−−− −55

ε67 ε48 ε46 ε74 ε97 ∑jεj

contribn to ΔE0

(in mV) −25.75 −14.75 −3.75 0.25 −5.75 −49.75

contribn to ΔE0

(in mV) for the

nitrated Tyr

−41.25 +14.75 −0.25 −26.25 −15.25 −68.25

distance to the

heme (Å) 6.5 10.2 9.4 12.6 14.0

Determined according to eq 1.

The Journal of Physical Chemistry B Article

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(−50 mV) to the redox potential shift of the null mutant of

−55 mV. This ﬁnding suggests that the main origin of the redox

potential shifts lies in a change of the polarity and electrostatics

of the protein environment rather than a mutation-induced

structural perturbation of the redox center. This conclusion is

consistent with the appreciable long-range eﬀect of the most

remote Tyr residue at position 97, although those residues that

are in closest proximity to the heme (i.e., at positions 67 and

48) exert the strongest inﬂuence on the redox potential.

The redox potentials determined in this work refer to the

native B1 state of the proteins immobilized on the MUA-coated

Ag electrode. Taking into account the interfacial potential drop

which leads to a small apparent negative shift of the redox

potentials,

the values for the WT protein and the null variant

are in good agreement with those in solution.

However, redox

potential shifts of the mono-Tyr mutants diﬀer slightly for the

protein in solution and on the electrode (Supporting

Information) which may be attributed to the distance- and

orientation-dependent modulation of εjby the interfacial

electric ﬁeld.

Eﬀect of Tyrosine Nitration on the Redox Center.

Upon nitration, a considerable fraction of the individual Tyr

variants is converted into states with non-native heme

coordination. Among them, a new 6cLS species is formed

with ca. 15% in all nitrated Cyt-cvariants except for Y97-NO2

(<10%). RR spectra indicate that this 6cLS species is closely

related the alkaline form of Cyt-cin which Met80 is replaced by

a Lys (72, 73, or 79). These ﬁndings are in agreement with

previous spectroscopic data on the nitrated Tyr mutants.

12−14

In WT Cyt-c, this alkaline species is formed at a pH above

9.5,

corresponding to the pKaof the surface-exposed lysine

residues. Thus, deprotonation of the Lys side chain is likely to

be the crucial step of this conformational transition and the

stronger binding aﬃnity of the Lys amino group to the ferric

heme compared to the thioether group of Met80 may

constitute a signiﬁcant thermodynamic driving force. However,

formation of the Lys/His coordinated species in the nitrated

Tyr mutants of Cyt-cat pH 7 seems to follow a diﬀerent

mechanism in view of the concomitant formation of the

prevailing 6cHS species which is attributed to a H2O/His heme

(Table 1). The coexistence of the 6cLS and the HS form

detected in the present RR spectroscopic study suggests that

the primary eﬀect of tyrosine nitration is the destabilization of

the native state B1, speciﬁcally the weakening of Fe−S(Met80)

bond, rather than a lowering of the pKaof one of the surface

lysines.

Nitration of the tyrosines does not display a uniform

tendency for the individual redox potential shifts of the B1 state

of immobilized protein (Table 3) which can be analyzed

according to eq 1. Whereas εjfor the nitrated Tyr at positions

67, 74, and 97 is distinctly more negative than for the non-

nitrated form, the value for ε46 remains small. Most strikingly,

however, there is a large positive shift for ε48 by 30 mV

compared to the non-nitrated Tyr. Note that in solution most

of the nitrated mono-Tyr mutants display a positive redox

potential shift compared to the non-nitrated forms.

Coordination State Changes and Peroxidase Activity.

Previous studies have shown that Tyr nitration leads to an

increase of the peroxidase activity.

7,13

Intuitively, one might

expect a direct correlation between the peroxidase activity and

the relative contribution of the HS species with its vacant (or

only weakly bound) axial coordination site, which increases in

the order Y48-NO2≈Y46-NO2> Y74-NO2≈Y67-NO2>

Y97-NO2(Table 1). The same order holds for the nitration-

induced destabilization of the native B1 state in general. For

Y46-NO2, Y48-NO2and Y74-NO2but not for Y67-NO2a

signiﬁcantly increased peroxidase activity was found.

13,14

Also,

the comparison with the enzymatic data for the site-speciﬁcally

nitrated wild-type Cyt-creported by Batthany et al.

does not

provide an unambiguous correlation with the present

spectroscopic results on the corresponding monotyrosine

mutants inasmuch as the peroxidase activity follows the order

Y74-NO2> Y67-NO2≈Y97-NO2. Accordingly, it appears to be

more plausible to assume that the peroxidase process is

primarily controlled by the accessibility of the heme pocket for

H2O2which requires a destabilization of the heme crevice.

This conclusion is in line with a previous UV RR study by Spiro

and co-workers.

These structural changes are accompanied by

a weakening of the Fe-Met bond which eventually may lead to

the removal of the ligand from the heme.

Eﬀect of the Electric Field on the Redox Site. The

relationship between destabilization of the heme crevice and

the distortion of the axial heme coordination has been observed

for Cyt-cbound to negatively charged liposomes including

cardiolipin or other anionic phospholipids,

23,25,26,44,45

or even

more simple membranes models such as an electrode coated by

a SAM with anionic head groups.

26−28,41

Thus, it has been

assumed that also electrostatic interactions, which govern Cyt-c

binding to the cardiolipin-rich inner mitochondrial membrane,

may promote the switch from the redox to the peroxidase

function.

In these electrostatic complexes, however, the

weakening of the Fe-Met leads to a 5cHS and a new non-native

6cLS state in which a His (33 or 26) replaces the Met80 ligand

of the heme iron (B2 states).

26−28

In fact, also the non-nitrated

and nitrated protein variants of human Cyt-cimmobilized on

SAM-coated electrodes show a similar behavior. The

component spectrum of the 6cLS species in the present

SERR spectra is essentially identical to that of the B2 6cLS state

of horse heart Cyt-c.

Conversely, the alternative assignment of

this species to the alkaline state is rather unlikely since the Lys

residues that might substitute the Met80 ligand are involved in

electrostatic binding of the protein to the SAM surface.

The extent of B2 formation, including both the 6cLS and the

HS forms, is higher for the human WT Cyt-cprotein than for

the WT Cyt-cfrom horse heart under similar experimental

conditions but it is further increased for the various Tyr

mutants, including the null mutant. For the non-nitrated

variants, the interfacial electric ﬁeld induces a decrease of the

B1 content by a factor of ca. 2. This factor is distinctly higher

than for the WT protein and the average value for the nitrated

Tyr mutants (1.3), except for Y97-NO2, such that the

contribution of the native B1 state has dropped to a similar

value of ca. 40% in all nitrated and non-nitrated mutants. These

ﬁndings suggest that the electric-ﬁeld-induced destabilization of

the redox site is largely independent of the site of the Tyr →

Phe substitutions and the Tyr nitration. In fact, the strength of

the electrostatic interactions and thus the electric ﬁeld

experienced by the bound proteins seem to remain largely

unchanged since the various Tyr →Phe substitutions and the

respective nitrations do not aﬀect the charge distribution in the

binding domain and do not signiﬁcantly alter the dipole

moment of the protein (Supporting Information).

The dipole moment of ferric human WT Cyt-chas been

calculated to be 254 D in the present work (Supporting

Information) and thus is distinctly higher than that of horse

WT horse heart Cyt-c(184 D),

implying that electrostatic

The Journal of Physical Chemistry B Article

dx.doi.org/10.1021/jp302301m |J. Phys. Chem. B 2012, 116, 5694−57025700

interactions on the MUA-coated Ag electrode are much

stronger for the human protein. One consequence refers to

the electric-ﬁeld-induced stabilization of the heme pocket which

is more severe for human WT Cyt-c(24% B2; Table 2) than for

horse heart Cyt-c(∼0% B2)

under the same immobilization

conditions. Consistently, destabilization of yeast iso-1 Cyt-c,

which exhibits a dipole moment 544 D in the ferric form,

even more severe than for human Cyt-c.Thesecond

consequence is related to the electron-transfer process in the

immobilized state which has been shown to be controlled by

the interplay between protein dynamics and electron

tunneling.

27,41,46,48

Since the thermodynamically preferred

binding domain of Cyt-cdoes not correspond to the orientation

that is optimum for electron transfer, a reorientation of the

bound protein must precede electron tunneling which is the

rate-limiting step for horse heart Cyt-con a MUA-coated Ag

electrode (kET =40s

−1). With increasing strength of

electrostatic binding, reorientation is slowed down and may

become the rate-limiting step as it is most likely the case for

WT human Cyt-c(kET =4s

−1; Table 3). For yeast iso-1 Cyt-c,

the strong electrostatic binding to the MUA surface even

impairs the spectroelectrochemical determination of the

interfacial electron transfer.

On the other hand, the increase

of the electron-transfer rate constant upon Tyr →Phe

substitution and Tyr nitration cannot be related to the

relatively small changes of the dipole moment by less than

10% compared to the WT protein (Supporting Information).

More detailed experimental and theoretical studies are required

to explore the impact of the mutational and chemical

modiﬁcations of Cyt-con the dynamics and the electron

tunneling pathways in the immobilized state.

■CONCLUSIONS

The eﬀect of Tyr nitration on the structural and redox

properties of the heme site of Cyt-cwas studied on the basis of

mono-Tyr mutants. This approach is based on the approx-

imation that the replacement of Tyr by Phe only causes minor

perturbations of the protein structure and the redox potential.

This assumption seems to be largely justiﬁed since crucial

structural parameters of the heme pocket are preserved,

including the sensitive Fe−Met axial bond. The RR

spectroscopic analysis reveals subtle conformational changes

beyond the level of heme ligand exchange, which are, however,

accompanied by redox potential shifts by up to −55 mV. Most

surprisingly, the eﬀects of the individual Tyr →Phe

substitutions on the redox potential are additive, pointing to

long-range electrostatic perturbations of the redox potential.

Nitration of the individual tyrosines causes more severe

structural changes of the heme pocket including a substantial

weakening of the Fe-Met axial bond which eventually leads to

partial formation of non-native ligation states. These states

include HS species and a Lys-His coordinated 6cLS species at

pH 7.0 with the same coordination pattern as the alkaline form

of the WT and the non-nitrated Cyt-cvariants formed above

pH 10.0. There is no direct correlation between the peroxidase

activity determined previously

7,13

and the relative contribution

of the HS forms or with the extent of coordination state

changes in toto. These ﬁndings support the view that nitration-

induced peroxidase activity is primarily due to the increased

accessibility of H2O2which results from the perturbation of the

heme crevice.

Upon electrostatic binding to SAM-coated electrodes, the

Fe−Met axial bond is weakened for both the nitrated and non-

nitrated protein variants to a comparable extent implying that

the electric-ﬁeld-induced destabilization of the heme pocket

structure is largely independent of the eﬀect of tyrosine

nitration. In the immobilized state, a Lys-His coordinated

species is not formed due to the involvement of Lys residues in

electrostatic binding to the SAM surface, but instead His33 (or

His26) evidently replaces Met80 to form a non-native 6cLS

state. The same structural changes, albeit not so pronounced,

are also observed for the WT protein.

Both Tyr nitration and membrane binding cause similar

albeit not identical destabilizations of the heme pocket, leading

to a decrease of the native B1 conformation. In both cases, the

non-native states, i.e., the HS forms, the alkaline-like species in

solution, and the His-His coordinated 6cLS form in the

immobilized state, display a strongly negatively shifted redox

potential

25,28,38,49

that impairs electron acceptance from

cytochrome creductase. Thus, the present results are consistent

with the view that either Tyr nitration or electrostatic binding

to the anionic cardiolipin-rich inner mitochondrial membrane,

or the combination of both events, may trigger the functional

switch from the redox to the peroxidase function of Cyt-c.

■ASSOCIATED CONTENT

SSupporting Information

The material includes a description of the RR and SERR

spectroscopic analysis, UV−vis absorption spectra, and details

of the MD simulations. This material is available free of charge

via the Internet at http://pubs.acs.org.

■AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +49-30-314-

21419.

Notes

The authors declare no competing ﬁnancial interest.

■ACKNOWLEDGMENTS

The authors thank the Spanish Ministry of Science and

Innovation (BFU2009-07190) and the Andalusian Government

(BIO198) for ﬁnancial support. The work was further

supported by the Cluster of Excellence “Unifying Concepts in

Catalysis”funded by the DFG.

■ABBREVIATIONS

CV, cyclic voltammetry; Cyt-c, cytochrome c; HS, high spin;

LS, low spin; MD, molecular dynamics; MUA, mercaptouna-

decanoic acid; RR, resonance Raman; SAM, self-assembled

monolayer; SERR, surface enhanced resonance Raman

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Unraveling the Interfacial Electron Transfer Dynamics of

Electroactive Microbial Biofilms Using Surface-Enhanced

Raman Spectroscopy

Ly, H. K.; Harnisch, F.; Hong, S.-F.; Schr¨

oder, U.; Hildebrandt, P; Millo, D. ChemSusChem

2013, DOI: 10.1002/cssc.201200626.

103

DOI: 10.1002/cssc.201200626

Unraveling the Interfacial Electron Transfer Dynamics of

Electroactive Microbial Biofilms Using Surface-Enhanced

Raman Spectroscopy

Hoang K. Ly,[a] Falk Harnisch,[b] Siang-Fu Hong,[b] Uwe Schrçder,[b] Peter Hildebrandt,[a] and

Diego Millo*[a, c]

Introduction

Microbial bioelectrochemical systems (BESs) are devices using

electroactive microbial biofilms that are attached to electrodes

for the catalysis of bioelectrochemical oxidations (at anodes)

and reductions (at cathodes). Microbial BESs are used for the

production of electricity and hydrogen, as well as for other ap-

plications.[1,2] BESs have an electroactive microbial biofilm on

the anode that relies upon an efficient electron transfer (ET)

strategy for delivering the electrons, gathered from the

oxidation of the microbial substrates, to the electrode.

The elucidation of ET mechanisms used by different micro-

organisms is motivating substantial research efforts.[3,4] Several

studies have reported active roles of soluble redox mediators,

membrane-bound redox proteins [that is, outer membrane cy-

tochromes (OMCs)], and conducting nanowires.[5,6] Although

all of these species appear to be involved in the ET process,

their mutual interplay and individual contributions to the ob-

served current densities are still unclear and under debate.[7,8]

These shortcomings are mainly attributable to the lack of ap-

propriate analytical methods for investigating the underlying

ET reactions in situ, that is, on living electrocatalytic microbial

biofilms.[9] The outstanding complexity of these microbial bio-

electrocatalytic processes, involving several electroactive spe-

cies that are difficult to probe individually, require an analytical

methodology providing selective information regarding the

redox and structural properties of the species promoting the

ET process. We have applied a spectroelectrochemical ap-

proach to gain insight into the kinetics of the ET that is selec-

tive for the biofilm/electrode interface. Whereas electrochemis-

try allows the monitoring and control of electrocatalytic activi-

ty and the redox processes of the biofilm in toto, spectroelec-

trochemistry based on surface-enhanced resonance Raman

(SERR) spectroscopy exclusively probes the heme groups of

the surface-confined OMCs (sc-OMCs) and their redox process-

es at the interface of the biofilm and the Ag electrode.[10] The

selectivity of the spectroscopic approach originates from the

near-field enhancement of the resonance Raman (RR) scatter-

ing of the heme groups that are in close vicinity (<7 nm)[11] to

the Ag working electrode. In contrast to other spectroelectro-

chemical approaches based on UV/Vis spectroscopy that probe

all of the heme groups across the entire biofilm (i.e., the outer

membrane and the periplasmic cytochromes over a distance

of tens of micrometers);[12] cytochromes other than sc-OMCs,

that is, cytochromes more distant from the electrode and

periplasmic cytochromes, do not contribute to the SERR

spectra. For the sc-OMCs, the SERR spectra provide information

on both the oxidation state and the structure (e.g., coordina-

tion and spin state) of the heme as well as the respective

changes during the redox processes of the biofilm. Moreover,

the method can be employed to probe the dynamics of these

processes by combining it with the electrochemical potential

The electron transfer (ET) processes of electroactive microbial

biofilms have been investigated by combining electrochemis-

try and time-resolved surface-enhanced resonance Raman (TR-

SERR) spectroscopy. This experimental approach provides se-

lective information on the ET process across the biofilm–elec-

trode interface by monitoring the redox-state changes of

heme cofactors in outer membrane cytochromes (OMCs) that

are in close vicinity (i.e., within 7 nm) to the Ag working elec-

trode. The rate constant for heterogeneous ET of the surface-

confined OMCs (sc-OMCs) of a mixed culture derived electroac-

tive microbial biofilm has been determined to be 0.03 s1.

In contrast, according to kinetic simulations the ET between

sc-OMCs and their redox partners, embedded within the bio-

film, is a much faster process with an estimated rate constant

greater than 1.2 s1. The slow rate of heterogeneous ET and

the lack of high-spin species in the SERR spectra rule out the

direct attachment of the sc-OMCs to the electrode surface.

[a] H. K. Ly, Prof. Dr. P. Hildebrandt, Dr. D. Millo

Institut fr Chemie, Sekr. PC14

Technische Universitt Berlin

Straße des 17. Juni 135, 10623 Berlin (Germany)

[b] Dr. F. Harnisch, Dr. S.-F. Hong, Prof. Dr. U. Schrçder

Institute of Environmental and Sustainable Chemistry

TU Braunschweig

Hagenring 30, 38106 Braunschweig (Germany)

[c] Dr. D. Millo

Biomolecular Spectroscopy/LaserLaB Amsterdam

Vrije Universiteit Amsterdam

De Boelelaan 1083, 1081 HV Amsterdam (The Netherlands)

E-mail: d.millo@vu.nl

Supporting Information for this article is available on the WWW under

http://dx.doi.org/10.1002/cssc.201200626.

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemSusChem 0000,00,1–7

These are not the final page numbers! ÞÞ

CHEMSUSCHEM

FULL PAPERS

step method. Here, the electrode potential is stepped from an

initial (Ei) to a final potential (Ef) to perturb the original redox

equilibrium at Ei. The subsequent relaxation processes towards

the equilibrium state at Efcan be monitored by measuring the

TR-SERR spectra at various delay times relative to the potential

step. This approach has successfully been applied to the study

of immobilized proteins.[13] Herein, we have applied this ap-

proach to electroactive microbial biofilms grown on Ag elec-

trodes to selectively obtain information regarding the ET re-

actions and possible coupled conformational transitions of sc-

OMCs. The biofilms are derived from mixed-culture inoculum;

the bioelectrocatalytic activity is dominated by Geobacteraceae

[14] In conjunction with electrochemical methods and by ex-

tending the approach to turnover and non-turnover condi-

tions, the study provides new insight into ET dynamics of

these biofilms.

Results

Under non-turnover conditions (i.e., in the absence of sub-

strate), the redox states of the sc-OMCs can be adequately

controlled by applying an appropriate potential to the biofilm-

covered working electrode.[10] To explore the ET dynamics of

the sc-OMCs, a potential step is applied from Ei=450 to Ef=

320 mV [vs. Ag/AgCl (3.0mKCl) reference electrode] to trig-

ger the transition from a largely reduced state of the sc-OMCs

(Ei) to a state of equal contributions of ferric and ferrous hemes

(Ef=E1/2) via ET from the sc-OMCs to the electrode. E1/2 can be

considered as the apparent equilibrium potential of sc-OMCs,

representing the average of the macroscopic redox potentials

of the individual OMCs that are centered at 280 and

360 mV (Figure S1).

TR-SERR spectra, recorded at different delay times after the

potential step, displayed an increase of the band at 1375 cm1

at the expense of the 1361 cm1band, which are characteristic

markers for the ferric and ferrous hemes in sc-OMCs, respec-

tively (Figure 1, upper panel). The TR-SERR spectra did not

reveal any indications of the involvement of intermediate

states other than the ferric and the ferrous forms in their

native bis-histidine ligated, six-coordinated, low-spin configura-

tions that were already assigned to a mixed-culture-derived

electroactive biofilm.[10] Furthermore, there were no time-de-

pendent changes of the overall SERR intensity during the po-

tential step experiments, which implied that the arrangement

of the sc-OMCs with respect to the electrode remained un-

changed (Figure S2). This finding excludes the potential-depen-

dent adsorption/desorption of sc-OMCs that has been suggest-

ed to explain the sharp cleft observed in the cyclic voltam-

metry (CV) trace of the Geobacteraceae species under non-turn-

over conditions, a feature that is also present in the CVs of the

biofilms in this study (Figure S1).[15,16]

The spectra obtained at different delay times were subse-

quently subjected to a component analysis to extract the rela-

tive contributions of oxidized and reduced sc-OMCs, expressed

as the molar fractions XOx and XRed, respectively.[10,17] The relaxa-

tion constant krelax =(0.060.04) s1, obtained from the fitting

of a single exponential function to the data, refers to the het-

erogeneous ET with zero-driving force (h=0 mV). The relaxa-

tion constant was found to be independent of the magnitude

of the potential step from Eito Ef(for Ef=E1/2) and was the

same irrespective of the direction of the step (Table S1), imply-

ing that oxidation and reduction of the sc-OMCs occurred at

the same rate. As krelax is equal to the sum of the rate constants

for oxidation (k1) and reduction (k2), we thus obtained k1=k2=

0.03 s1(vide infra). It should be noted that TR-SERR experi-

ments carried out with shorter delay times rule out any tran-

sient changes of sc-OMCs on the millisecond time scale (Fig-

ure S3). Furthermore, an increase in the driving force by select-

ing jEfj>jE1/2 j(h¼6 0 mV) has no impact on the relaxation

constant determined by TR-SERR spectroscopy (Table S1).

Static potential-dependent SERR spectra obtained under

turnover conditions (i.e., in the presence of substrate) showed

that the majority of the sc-OMCs are reduced, independent of

the oxidizing electrode potential (Figure S4). Approximately

only 10% of the heme cofactors remained oxidized throughout

the entire potential window investigated. As a 10% fraction of

sc-OMCs was shown to be redox-inactive under non-turnover

conditions,[10] we concluded that such biofilms inevitably in-

clude a small portion of sc-OMCs that, possibly because of an

unfavorable orientation, do not participate in electron

exchange with the electrode.

Figure 1. Upper panel: selected TR-SERR spectra of the biofilm under non-

turnover conditions obtained at delay times of 9, 37, and 67 s after the ap-

plied potential step from Ei=450 mV to Ef=320 mV. Lower panel: time-

dependent changes of the mole fractions of reduced (&) and oxidized (*)

sc-OMCs determined from the TR-SERR spectra, measured under non-turn-

over conditions, following a potential step from Ei=450 mV to

Ef=320 mV. The solid lines refer to the mono-exponential fits to the exper-

imental data.

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TR-SERR experiments under turnover conditions, carried out

with a wide dynamic range from a few milliseconds to

a number of seconds, indicate that there are no transient

changes of the oxidation state of the sc-OMCs (Figure S5).

The applied potential steps were also monitored by

chronoamperometric measurements to record simultaneous

spectroscopic signals and corresponding current (i) versus time

(t) traces. Because the entire biofilm is conductive, the current

flow reflects a superposition of all charge-transfer processes

originating from all layers of the biofilm.[18] Separating the ob-

served current into the different individual contributions (i.e.,

surface-confined and more remote OMCs, periplasmic cyto-

chromes, conducting nanowires, and non-Faradaic processes)

requires a profound knowledge of the architecture of the bio-

film. Because of the lack of this information, we were unable

to separate the current flow with respect to redox processes

that were not directly probed by SERR spectroscopy. The

iversus ttraces obtained under non-turnover conditions dis-

play biphasic behavior, showing an initial current flow leveling

off after a few seconds, followed by a slower relaxation phase

(Figure S6). Accordingly, each iversus ttrace has been fitted to

a biexponential function, affording kfast >1s

1and kslow =

(0.040.02) s1for the fast and the slow process, respectively.

The rate constant of the slow process is in very good agree-

ment with the heterogeneous ET rate constants derived from

the TR-SERR experiments (Table S2 and S1, respectively).

Conversely, the fast process cannot be ascribed to redox

charging/discharging processes of the sc-OMCs. Such an as-

signment is highly unlikely as no changes of the oxidation

state of sc-OMCs could be detected by TR-SERR spectroscopy,

even on the millisecond time scale. Thus, the fast component

of the amperometric response to the potential step must refer

to another unidentified charge-transfer process in the biofilm.

Discussion

The TR-SERR spectroscopy experiments under non-turnover

conditions showed that heterogeneous ET across the sc-OMCs/

electrode interface was a slow process. No oxidized sc-OMCs

were transiently formed under turnover conditions, which

implied that there was a rapid reduction of the sc-OMC via

a fast ET from a pool of redox partners, assigned to a bulk

redox center (b-RC) that was more remote from the metal sur-

face and thus, in contrast to sc-OMCs, was not visible in the

SERR spectra.

Accordingly, the experimental kinetic data obtained in this

study could be explained by using a simple model that as-

sumed a pool of two redox centers, that is, sc-OMCs and a b-

RC (Figure 2). The sc-OMC was directly monitored by SERR

spectroscopy and was characterized by (i) an average redox

potential that defined the equilibrium between the reduced

and oxidized species, and (ii) a rapid electron exchange be-

tween the heme groups within the sc-OMC. The b-RC was not

directly accessible using SERR spectroscopy, but it was as-

sumed to exchange electrons with the sc-OMC. As all of the

microbial cells within the biofilm were electrically wired to the

electrode, there must have been a redox species, such as b-RC,

that provides such an electrical contact. The lack of substantial

information regarding the protein architecture of model

Geobacteraceae biofilms in close vicinity to the electrode

(i.e.,<7 nm) means that the nature of the b-RC cannot be

identified. However, according to the models describing the ET

process within Geobacteraceae biofilms, b-RC could be an OMC

(as recently proposed by Bonanni et al.),[19] a conducting nano-

wire, or a periplasmic cytochrome. Regardless of the nature of

b-RC, the kinetic description was restricted to the heteroge-

neous ET (k1=k2, vide supra) of the sc-OMC proteins to (and

from) the electrode and the ET between the two pools (k3and

k4).

We first considered the TR-SERR experiment under turnover

conditions using a potential step from 450 mV to 0 mV.

At Ei=450 mV the biofilm was catalytically inactive and the

pool of sc-OMCs was fully reduced. After the potential was

stepped to 0 mV, no oxidized sc-OMC species were detectable

even on the millisecond time scale; therefore, we concluded

that ET from the b-RC to the sc-OMC pool was much faster

than the heterogeneous ET, that is, k4@k1=k2.

It is reasonable to assume that this relationship was also

true for non-turnover conditions and thus for the description

of the TR-SERR data in Figure 1. According to the model, the

ratio between k3and k4depends upon the difference between

the equilibrium potentials of b-RCs and sc-OMCs, which is not

known a priori. We therefore carried out kinetic simulations for

various k4/k3ratios on the basis of k4@k1=k2. The simulations

(Figure 3) only provided a good description of the experimen-

tal data in Figure 1 when k3k4(further details are given in

Text S1). This finding implies that the equilibrium potentials of

the b-RCs and sc-OMCs were very similar (Text S2). Under the

assumption that each cytochrome of the sc-OMC pool ex-

changed electrons with one redox partner of b-RC, the ET pro-

cess between the pools had to be at least 40 times faster than

the heterogenous ET between the sc-OMC and the electrode

Figure 2. Representation of the scheme adopted to describe the ET across

the bacteria/electrode interface and within the microbial biofilm. The red

dots on the left panel represent the heme groups of the sc-OMCs. These sc-

OMCs exchange electrons with the electrode (in gray), characterized by the

rate constants k1and k2, and with the b-RCs, described by the rate constants

k3and k4. The black arrow represents the route followed by the electrons in

a reduction process. In the right panel, the sc-OMC depicted on the left

panel is represented by the average equilibrium potential of the correspond-

ing OMC (i.e. E1/2).

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surface. Therefore, we tentatively estimated the rate constant

k4to be larger than 1.2 s1.

The slow rate of heterogeneous ET ruled out the direct at-

tachment of the redox-active sc-OMCs to the Ag surface. This

conclusion is consistent with the lack of high-spin heme spe-

cies in the SERR spectra that are usually observed for cyto-

chromes adsorbed on bare Ag electrodes.[20,21] Conversely, the

sc-OMCs needed to be separated from the electrode by using

poorly conducting biomaterials up to a distance that account-

ed for the slow ET rate but was sufficient for an appreciable

surface enhancement of the RR signals. Considering heteroge-

neous ET rates and the SERR intensities observed for heme

proteins located at well-defined distances from the elec-

trode,[22] the average heme-to-electrode distance of sc-OMCs

was in the range of 3–5 nm. At such long distances, direct elec-

tron tunneling from the heme to the electrode was less proba-

ble and the lack of an overpotential-dependence indicated

that the heterogeneous ET must be controlled by a different

mechanism. A plausible explanation for these findings could

be an electron hopping mechanism as suggested previously

for similar slow and overpotential-independent ET over compa-

rably long distances.[23,24] Alternatively, it can be assumed that

the heterogeneous ET is limited by another process, tentatively

ascribed to proton transfer or biofilm charge/discharge

attributable to ion mobility.[4,5]

In conclusion, the lack of an appreciable steady-state or tran-

sient concentration of oxidized sc-OMCs in the static and TR-

SERR spectra under turnover conditions was because of slow

heterogeneous ET, which is likely to be dependent upon the

way in which the sc-OMCs are linked to the metal surface. Our

conclusions differ substantially from the currently accepted

model of the ET within microbial biofilms.[15,19] This model com-

prises several steps, each one accounting for a different ET

process through the entire biofilm and it has been tested with

electrochemical techniques on pure Geobacteraceae species.

Consistently, two groups have estimated very large heteroge-

neous ET rate constants of 5000 and 10000 s1. Although the

sub-millisecond timescale is accessible for TR-SERR spectrosco-

py, no redox activity associated to the sc-OMCs has been ob-

served on that timescale in our study (see the Supporting In-

formation). Based on this model, Bond et al. predicted a con-

centration gradient of oxidized cytochromes inside the biofilm

under turnover conditions, with cells closer to the anode

having the highest concentration of oxidized OMCs.[25] These

findings have recently been supported by confocal resonance

Raman spectroelectrochemical analysis.[26] Although the pres-

ence of such a gradient in the bulk biofilm cannot be directly

probed by SERR spectroscopic measurements, the present TR-

SERR data under turnover conditions clearly showed that the

large majority of sc-OMCs is reduced. These discrepancies may

arise from the different electrode material (Ag vs. carbon), the

different growth conditions (mixed vs. pure cultures and differ-

ent applied potentials), or the interplay between these two as-

pects (e.g. different electrode materials may select different

bacterial communities) as well as the presence of further heter-

ogeneous ET mechanisms that could not be detected in our

experiments. Accordingly, it would be premature to generalize

our conclusions to different biofilm systems.

The present study does not prove nor disprove the conclu-

sions regarding the rate-limiting step of catalytic current pro-

duction identified in the ET from acetate to internal cyto-

chromes.[15,19] Our results have shown that the slow interfacial

ET promoted by the sc-OMCs is followed by a faster, not yet

assigned ET process. The present results support the hypothe-

sis that the sc-OMCs act as gates for the electrons exchanged

between the electrode and the bulk biofilm, as recently pro-

posed by Inoue et al.[27] Importantly, this model does not ex-

clude the presence of different ET strategies. The b-RCs depict-

ed in Figure 2 could also exchange electrons directly with the

electrode through conductive nanowires. However, when the

direct ET between these centers occurs (as proven by the pres-

ence of a layer of reduced sc-OMCs under turnover conditions),

this process is gated by the slow interfacial ET, as shown in the

model depicted in Figure 2.

Conclusions

This first TR-SERR spectroscopy study on electroactive microbi-

al biofilms grown on Ag working electrodes has provided un-

precedented information regarding the kinetics of the microbi-

al ET. The dynamics of the ET across the biofilm/electrode inter-

face has been directly probed by monitoring the heteroge-

neous ET of the sc-OMCs. The kinetic simulation of the TR-SERR

spectroscopy data obtained under turnover and non-turnover

conditions indicates a slow heterogeneous ET of the sc-OMCs,

which in turn undergoes rapid electron exchange with a b-RCs.

Because of the slow heterogeneous ET, which may reflect an

electron hopping mechanism, the sc-OMCs serve as gates for

the electrical coupling of the biofilm with the electrode. In this

sense, the TR-SERR spectroelectrochemical approach has been

shown to be a powerful tool for selectively probing the inter-

Figure 3. Plot of the difference between the root mean square deviations

RMSexp and RMSsim versus the rate constant ratio k3/k2(&). RMSsim refers to

the deviation of the kinetic simulation (according to Figure 2) with respect

to the experimental data (Figure 1), using k1=k2=0.03 s1and k4=1.2 s1

(see text and Text S2 for further details). RMSexp represents the reference

value of the mono-exponential fit to the experimental data in Figure 1, de-

termined to be 0.990. The relationship between RMSexp–RMSsim and k3/k2can

be approximated by a mono-exponential function (solid line). The k3/k2ratio

of 7.2 that corresponds to RMSsim =RMSexp (dashed line) thus affords the

lower limit for k3, that is, k30.22 s1.

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facial processes of biofilms close to the electrode surface, both

under turnover and non-turnover conditions. Furthermore,

these findings may also allow for the elucidation of mecha-

nisms for the recently discovered interspecies ET, for example,

in activated sludge granules.[28]

Experimental Section

Microbial inoculum and growth medium

The source of the microbial inoculum was primary waste water,

collected from the waste water treatment plant (WWTP) Braun-

schweig/Steinhof, Germany. The bacterial growth medium con-

tained NH4Cl (0.31 gL1), KCl (0.13 gL1), NaH2PO4·H2O (2.69 gL1),

Na2HPO4(4.33 gL1), trace metal (12.5 mL), and vitamin (12.5 mL)

solutions according to Kim et al.[29] Acetate solution (10 mm,

pH 6.8) served as the substrate in the growth medium. All

solutions were purged with nitrogen before use.

Primary and secondary biofilm formation

As described by Liu et al.[30] for the formation of primary biofilms,

1 mL of waste water per 30 mL of the substrate solution was in-

oculated into a sealed electrochemical cell incubated at 358C.

A constant potential of 200 mV versus Ag/AgCl (3.0mKCl) was ap-

plied to the working electrode [graphite rods (CP-Graphite GmbH,

Germany)] using a potentiostat (Potentiostat/Galvanostat VMP3,

BioLogic Science Instruments, France) to promote and monitor the

biofilm formation. The biofilm growth was monitored by measur-

ing the bioelectrocatalytic oxidation current and the substrate (ace-

tate) consumption was analyzed by using HPLC. The exhausted

substrate solution was replenished regularly until a steady-state

maximum current density was achieved (usually after 3–4 feeding

cycles). Afterwards, the primary biofilm was scraped from the

carbon electrode in anaerobic and sterile conditions and was used

as bacterial inoculum for the secondary biofilm formation following

a similar procedure on Ag ring disc electrodes at the applied po-

tential of 50 mV versus Ag/AgCl (3.0mKCl).[30] After biofilm for-

mation (j600 mAcm2, Figure S 8), these electrodes were used for

TR-SERR spectroscopy experiments as described below.

Spectroelectrochemical set-up

Electrochemical measurements on the microbial biofilm were car-

ried out in a homemade spectroelectrochemical cell working in the

three-electrode configuration and controlled by a mAutolab poten-

tiostat (Eco Chemie, Utrecht, The Netherlands). The working elec-

trode was a Ag ring with a projected surface area of 0.7 cm2. All

current densities are expressed with respect to the projected elec-

trode area. After mechanical polishing, the electrode was rough-

ened by using the electrochemical procedure described else-

where,[31] and transferred to the electrochemical cell for biofilm

growth (vide supra). A Pt coil and a Ag/AgCl (3.0mKCl; Dri-Ref,

WPI Berlin, Germany) served as the counter and the reference elec-

trode, respectively. All potentials provided in the manuscript are re-

ferred to the Ag/AgCl (3.0mKCl) reference electrode (210 mV

versus SHE).

SERR spectroscopy measurements

SERR spectra were measured using a confocal Raman spectrometer

(LabRam HR 800, Jobin Yvon) coupled to a liquid-nitrogen-cooled

charge-coupled device (CCD) detector. The spectral resolution was

1cm

1with an increment per data point of 0.75 cm1. For laser ex-

citation, the 413 nm line of a Krypton laser (Coherent Innova 400)

was used. The laser power on the sample was 1.0 mW. The laser

beam was focused onto the sample by using a Nikon 20x objective

with a working distance of 20.5 mm and a numeric aperture of

0.35. Acquisition times of the SERR spectra ranged between 5 and

60 s. The working electrode was constantly rotated to avoid laser-

induced sample degradation. All SERR measurements were carried

out in 30 mmphosphate buffer solution at pH 7.

Time-resolved measurements followed the previously described

procedure.[31,32] Briefly, potential steps were coupled with short

SERR measuring intervals that were defined by gating the continu-

ous-wave laser beam via electrooptical intensity modulators.

A home-made multi-channel delay generator was coupled to the

potentiostat (EG&G Princeton Applied Research, model 263 A) and

the intensity modulators (Linos M 0202) to synchronize potential

steps with the measuring intervals.

Acknowledgements

The work was supported by the DFG (Cluster of Excellence

“UniCat”, PH; SFB 803, CS), the Alexander-von-Humboldt founda-

tion (DM) and the Netherlands Organisation for Scientific Re-

search (NWO) grant 722.011.003 (DM). F.H. acknowledges support

by the Fonds der Chemischen Industrie (FCI). U.S. acknowledges

the foundation of the professorship Sustainable Chemistry and

Energy Research by the Volkswagen AG and the Verband der

Deutschen Biokraftstoffindustrie e.V. We would like to thank the

reviewers for the thoughtful observations that improved the qual-

ity of our work.

Keywords: cytochromes ·electron transfer ·kinetics ·

microbial fuel cells ·Raman spectroscopy

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Received: August 23, 2012

Revised: November 5, 2012

Published online on &&

, 0000

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FULL PAPERS

H. K. Ly, F. Harnisch, S.-F. Hong,

U. Schrçder, P. Hildebrandt, D. Millo*

&& –&&

Unraveling the Interfacial Electron

Transfer Dynamics of Electroactive

Microbial Biofilms Using Surface-

Enhanced Raman Spectroscopy

Speed dating for electrons: Heteroge-

neous electron transfer across the bio-

film/electrode interface is a slow pro-

cess promoted by surface-confined cy-

tochromes that are not in direct physi-

cal contact with the electrode. Subse-

quent electron transfer from the

surface-confined cytochromes to more

remote redox centers within the biofilm

is a much faster process. Herein, an ap-

proach is identified as a powerful tool

for selectively probing interfacial pro-

cesses of biofilms close to the electrode

surface.

 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemSusChem 0000,00,1–7

These are not the final page numbers! ÞÞ

Induced Surface Enhancement in Coral Pt Island Films

Attached to Nanostructured Ag Electrodes

Ly, H. K.; K¨

ohler, C.; Fischer, A.; Kabuß, J.; Schlosser, F.; Schoth, M.; Knorr, A.;

Weidinger, I. M. Langmuir 2012, 28, 5819–5825.

113

Induced Surface Enhancement in Coral Pt Island Films Attached to

Nanostructured Ag Electrodes

H. Khoa Ly,

†

Christopher Kohler,

‡

Anna Fischer,

†

Julia Kabuss,

‡

Felix Schlosser,

‡

Mario Schoth,

‡

Andreas Knorr,

‡

and Inez M. Weidinger*

,†

†

Institut fur Chemie, Technische Universitat Berlin, Strasse des 17 Juni 135, D-10623 Berlin, Germany

‡

Institut fur Physik, Technische Universitat Berlin, Hardenbergstrasse 36, D-10623 Berlin, Germany

SSupporting Information

ABSTRACT: Coral Pt islands films are deposited via

electrochemical reduction on silica-coated nanostructured Ag

electrodes. From these devices surface-enhanced (resonance)

Raman [SE(R)R] signals of molecules exclusively attached to

Pt are obtained with intensity up to 50% of the value

determined for Ag. SE(R)R spectroscopic investigations are

carried out with different probe molecules, silica-coating

thicknesses, and excitation lines. Additionally, field enhance-

ment calculations on Ag−SiO2−Pt support geometries are

performed to elucidate the influence of the Pt island film

nanostructure on the observed Raman intensities. It is concluded that the nonperfect coating of the Pt island film promotes the

efficiency of the induced Pt SER activity. Comparison with similar measurements on Ag−SiO2−Au electrodes further suggests

that the chemical nature of the deposited metal island film plays a minor role for the SE(R)R intensity.

1. INTRODUCTION

Surface chemistry plays a key role in many fields of modern

technology such as heterogeneous catalysis, solar energy

conversion, or bioelectronics. The efficiency of the surface-

confined processes sensitively depends on the interaction of

adsorbates with their underlying support. Hence, changing the

chemical nature and surface morphology of the support as well

as the properties of the surrounding medium can greatly

enhance or diminish the performance of the device. Therefore,

considerable research efforts have been made to design new

types of support materials. However, probing the adsorbates

and their processes on such surfaces represents a considerable

challenge since there are no generally applicable analytical

techniques that provide information about the molecular

structure and dynamics of the adsorbates under in situ

conditions.

In contrast to most surface-sensitive methods that are only

applicable to solid/gas interfaces at very low pressures, surface

enhanced Raman (SER) spectroscopy can be employed to

surfaces irrespective of the kind of surrounding medium and

thus may be the in situ analytical method of choice in surface

chemistry.

1−5

The main drawback of SERS that currently

prevents a wider applicability is related to the signal

enhancement mechanism which requires the resonant coupling

of radiation with surface plasmons of the metallic support. Such

plasmon resonances strongly depend on the dielectric function

and surface morphology of the metal, and so far mainly Ag and

Au have been demonstrated to be capable of providing

sufficient surface enhancement for reliable SER analysis.

particular, Ag affords a strong surface enhancement in a wide

spectral range from the violet to the near-infrared region,

7−9

but unfortunately this metal is only of minor interest for many

technological applications. In this respect, other metals such as

Pt or Pd are much more relevant; however, their intrinsic SERS

activity is lower by several orders of magnitude than in the case

of Ag

such that they are not considered as suitable supports

for in situ SER spectroscopy.

To overcome this drawback several approaches have been

developed in the past to induce SER activity for molecules

adsorbed on plasmonic inactive surfaces. All these approaches

have in common that they are based on hybrid systems

consisting of a plasmonic component such as Ag or Au for

optical amplification and a nonplasmonic component for

surface chemistry: In tip-enhanced Raman (TER) spectroscopy

surface enhancement is provided by a nanoscaled Au(Ag) tip

that is brought in close vicinity to the adsorbate on a

nonplasmonic support.

11−13

Although much insight can be

given by this technique it requires a rather demanding setup.

In-situ spectroscopy, however, does not necessarily need the

high spatial resolution of TER spectroscopy but relies upon a

flexible setup that can be easily adapted to specific applications

such as heterogeneous catalysis in reactors or electrochemical

cells in bioelectronics. Therefore, alternatively a strategy was

proposed where the tip is replaced by an ensemble of

nanoparticles that are spread onto the probed surface.

14−16

This approach avoids the sophisticated TER setup but at the

Received: December 29, 2011

Revised: February 13, 2012

Published: March 8, 2012

Article

pubs.acs.org/Langmuir

same time largely decreases the available area for surface

chemistry.

Finally, an overlay approach was introduced by Weaver et al.

and further developed by us and others where surface

enhancement is induced into a nonplasmonic metal film by

an underlying nanostructured SERS active support.

17−23

In our

previous work, electrochemically roughened Ag electrodes were

used as SERS active support and Au as an outer metal layer for

surface chemistry. The induced surface enhancement experi-

enced by molecules attached to Au was measured under violet

light excitation where Au itself does not show intrinsic

plasmonic activity. In contrast to the strategy of Weaver et

al., we introduced a defined separation of Au from the rough Ag

electrode by coating the latter with a dielectric spacer of defined

thickness. Repellent functionalization of the spacer layer

guaranteed exclusive adsorption of the adsorbates under

investigation on Au even for a nonperfectly closed metal film.

With this strategy the enhancement of Raman signals of the

probe molecule, i.e., the heme protein cytochrome c (Cyt-c),

was found to be similar on the outer Au layer as for direct

immobilization on the Ag support. This highly unusual long-

range enhancement over a spacer thickness of more than 20 nm

was rationalized by field enhancement calculations of Ag−

spacer−Au devices.

In this paper we successfully expanded our approach to coral

Pt island films. By variation of the excitation line, target

molecules, and spacer thickness, it was possible to identify

crucial parameters for optimum induced surface enhancement

at plasmonic inactive metal films. In addition, field enhance-

ment calculations were performed on different multilayer Ag−

spacer−Pt geometries to rationalize the experimental results.

2. MATERIALS AND METHODS

2.1. Chemicals. Tetrachloroplatinate(II) acid (H2[PtCl4]·6H2O,

99.9%), tetraethyl orthosilicate(TEOS,99.99%),aminopropyl

triethoxysilane (APTES), 3-mercaptopropylmethyldimethoxysilane

(MPTS), 11-amino-1-undecanethiol (AUT), and 11-mercapto-un-

decanoic acid (MUA) were purchased from Sigma Aldrich. Ethanol

(99.99%), isopropyl alcohol (99%), and ammonium hydroxide (35%

aqueous solution) were obtained form Fischer Scientific Co.

(Germany). Potassium hydrogen phosphate and potassium dihydro-

gen phosphate were provided by Merck (Germany). Benzenethiol

(BT, 98%) and mercaptopyridine (mPy, > 95%) were purchased from

Sigma-Aldrich. All reagents were of analytical grade and used as

received. Solutions were prepared using ethanol of analytical grade

(99.99%) or Millipore water (Eschborn, Germany) with a resistance >

18 MΩ. Ag ring electrodes of 8 mm diameter and 2.5 mm height were

machined from 99.99% Ag rods (Goodfellow, U.K.). Cytochrome c

(Cyt c, Sigma-Aldrich) was purified as described previously.

2.2. Spectroscopic and Electrochemical Measurements.

Spectroelectrochemistry was performed using cylindrical Ag and

Ag−S−Pt rings as working electrode, an Ag/AgCl (3 M KCl)

reference electrode (+0.21 V vs SHE), and a platinum counter

electrode. SER(R) spectra were measured using a confocal Raman

spectrometer (LabRam HR 800, Jobin Yvon) coupled to a liquid-

nitrogen-cooled CCD detector. The spectral resolution was 1 cm−1

with an increment per data point of 0.28 and 0.15 cm−1using the 413

and 514 nm laser excitation line, respectively. For laser excitation the

413 nm laser line of a Krypton (Coherent Innova 300c) or the 514 nm

line of an argon cw laser (Coherent Innova 70c) was used. The laser

power on the sample was 1.0 mW. The laser beam was focused onto

the sample by a Nikon 20×objective with a working distance of 20.5

mm and a numeric aperture of 0.35. Accumulation times of the SERR

spectra were between 1 and 12 s. The working electrode was

constantly rotated to average over a high electrode surface and to

avoid laser-induced sample degradation. All SER(R)S measurements

were done in 30 mM PBS buffer solution. Cyclic voltammetric

experiments were performed with a CH instrument 660 C (Austin,

USA).

2.3. Electrode Characterization. The surface morphology and

elemental composition of the electrodes was characterized with

scanning electron microscopy (SEM) and energy-dispersive X-ray

spectroscopy (EDX) using a JEOL 7401F operated between 8 and 12

kV equipped with an EDX detector Quantax XFlash Detektor 4010

from Bruker. Specific surface area measurements using multipoint BET

were done with Krypton gas adsorption measurements at 77.4 K with

an Autosorb-1-C from Quantochrome. Samples were degassed at 80

°C overnight prior to measurement.

2.4. Theoretical Calculations. The field distribution was

simulated using the Maxwellsolver JCMsuite, a finite element software

for computation of electromagnetic waves, developed by the Zuse

Institut Berlin.

The experimental structures were modeled by a

three-dimensional but rotationally symmetrical geometry. An external

Figure 1. Schematic presentation of the Ag−SiO2−Pt electrode preparation. First, an Ag electrode was electrochemically roughened, followed by

coating with a dielectric SiO2spacer layer of defined thickness. Finally, a nanostructured Pt island film was electrochemically deposited on top of the

spacer. (Bottom) SEM pictures of Ag−SiO2(A) and Ag−SiO2−Pt (B) electrodes, and an EDX spectra of the Ag−SiO2−Pt electrode (C).

Langmuir Article

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electromagnetic field was applied with defined polarization, wave-

length, and vector amplitude to calculate the field enhancement.

3. RESULTS

3.1. Electrode Preparation. Ag−SiO2−Pt electrodes were

created following the procedure established for Ag−SiO2−Au

in our previous work

19,21

with some modifications. The steps

for electrode preparation are illustrated in the top half of Figure

1. First, a nanostructured Ag support was created from a

cylindrical Ag bulk electrode via electrochemical roughening

using an established protocol.

27,28

The rough Ag electrode was

then coated with a silica layer of variable thickness. Thin layers

(0.7 nm) were formed by self-assembly of MPTS in ethanolic

solution.

21,29

Thicker SiO2layers were generated by additional

incubation of the electrode in a TEOS solution for 2 h.

The

thickness of the SiO2layer could thereby be tuned by adjusting

the concentration of the TEOS precursor.

Subsequently, the

Ag−SiO2electrode was dipped in APTES solution overnight to

obtain a positively charged amino-functionalized surface. This

step resulted in an additional layer thickness of 1.0 nm.

32,33

An SEM picture of an Ag−SiO2electrode surface is shown in

Figure 1A. It can be clearly seen that a closed and dense SiO2

film covers completely the Ag surface. The average thickness of

the SiO2layers can be estimated directly from the picture using

the included scale. The values were then correlated with the

TEOS concentration in the precursor solutions. The thickness

of the SiO2film in Figure 1A was determined to be 15 nm

corresponding to an initial TEOS concentration of 6 μM. In the

case of MPTS−APTES-coated electrodes the layer thickness of

1.7 nm was estimated by adding the respective lengths of the

individual monolayers. It should be noted that attaching SiO2

does not affect the surface morphology of the electrode. If the

surface pattern of the Ag electrode is approximated by an

arrangement of connected coral spheres an average coral

diameter of d=85±20 nm can be estimated. In the last step,

the Ag−SiO2electrode was dipped into an ethanol solution of 2

wt % of H2[PtCl4]·6H2O for 30 min to allow for electrostatic

binding of PtCl4

−ions to the positively charged amino groups

of the silica surface. After changing the buffer solution, a

potential of −0.5 V was applied which resulted in a reduction of

[PtCl4]2−ions and a Pt island film was generated on top of the

Ag−SiO2electrode. In Figure 1B an SEM picture of the

electrode after electrochemical Pt deposition is shown. The

electrode surface is now covered by a Pt island film that exhibits

also a coral-like structure but with a smaller coral size than the

underlying Ag support. The average diameter of the Pt

nanocorals is approximated by 30 ±10 nm. The presence of

Pt could be proven by EDX measurements that can be seen in

Figure 1C. It has to be noted that with increasing incubation

time of the Ag−SiO2-coated electrode in the PtCl4solution also

islands with a thicker, film-like morphology were formed upon

Pt reduction (see Figure S1 Supporting Information). Thicker

and thus more closed films, however, led to a decrease in SER

intensity of adsorbates (vide infra).

3.2. Determination of REF for Rough Ag Electrodes.

Prior to estimation of the surface enhancement of Ag−SiO2−Pt

electrodes the Raman enhancement factor (REF) of the pure

electrochemically roughened Ag electrodes had to be

determined according to eq 1

=· =·

Γ·

REF SERS

SERS

SERS (1)

where IRand ISERS are the Raman intensities of probe molecules

in solution and adsorbed on the Ag surface, respectively. These

intensities are normalized to the same accumulation time and

laser intensity. NRand NSERS refer to the number of molecules

that are in the focus of the laser beam. These quantities are

related to the product of illuminated volume Vand bulk

concentration cRin the normal Raman and to the product of

the illuminated area Aand the surface concentration ΓSERS in

the SER experiments. Benzenethiol (BT) was used as a Raman

probe to estimate REF following the procedure described by

McFarland et al.

Raman and SER spectra were measured for

BT in solution and adsorbed on the surface using 413 and 514

nm excitation. The intensity ratio of ISERS/IRwas subsequently

determined for the 1000 and 1080 cm−1line. The average ratio

considering both bands is shown in Table 1. The concentration

of the neat BT solution was cR= 9.54 mol/L. The irradiated

volume Vwas approximated by a cylinder with radius rand

height h

=π· ·Vrh

(2)

where ris given by the radius of the laser focus which has been

estimated by McFarland et al. to be r=2μm for a 20×

objective independent of the laser wavelength. The height of

the cylinder can be approximated by the depth of focus of a

Gaussian beam

=··

hr22

(3)

Accordingly, the surface area probed in the SERS measure-

ments is given by

=· =·π·

RF A RF r

geom 2

(4)

where Ageom stands for the geometrical area of the electrode

illuminated by the laser. To obtain the real surface area one has

to multiply by a factor that accounts for the surface roughness

of the electrode. The surface roughness factor (RF) was

determined independently by BET measurements to be 20.

The surface concentration of ΓSERS = 1.1 nM/cm2of BT on Ag

was taken from ref 34.

On the basis of these parameters, we obtain a value for REF

=2×103for 514 nm and 5 ×102for 413 nm excitation. These

values have to be seen as lower limits as we assume that all parts

of the surface are accessible by laser light and that a compact

BT monolayer is formed on the surface. Nevertheless, the low

REF values can be rationalized by the fact that, due to the

random coral structure, a large fraction of the surface plasmons

is not in resonance with the incoming light. Hence, the

averaged signal that is observed in the SERR spectrum might

originate only from a small percentage of the surface area. In

return this heterogeneity ensures surface enhancement over a

broad range of excitation lines.

3.3. SER Spectroscopy of mPy on Pt. Mercaptopyridine

(mPy) was used as a Raman probe to test surface enhancement

at the Pt surface in Ag−SiO2−Pt hybrid electrodes. The

Table 1. Ratio between SERS and Normal Raman Intensities

of BT and Corresponding Raman Enhancement Factors

(REF) of Electrochemically Roughened Ag Electrodes

λ/nm ISERS/IRREF

514 1.1 2 ×103

413 0.2 5 ×102

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different electrodes were incubated in a 5 mM mPy aequeous

solution for 20 min. SER spectra were measured in pure buffer

solution after rinsing and incubation in 1 M KCl for 10 min to

remove the loosely bound mPy. A typical SER spectrum of mPy

adsorbed on bare Ag at 514 nm excitation is shown in

Figure 2A. SER measurements under the same conditions using

an MPTS−APTES-coated Ag electrode yielded a more than 20

times lower signal intensity of mPy. If, however, the electrode

was coated additionally with a Pt island film the mPy intensity

increased again to roughly 50% of the Raman intensity

measured on bare Ag (Figure 2A, top). The intensity of the

1096 cm−1band of mPy adsorbed on Ag was ascribed to the

Raman enhancement factor (REF) of 2 ×103(vide supra).

Correspondingly, REF for mPy on Pt was derived by

comparison of this band’s relative intensity with respect to its

value on Ag. The values for log(REF) obtained in this way are

plotted in Figure 2B for two different spacer thicknesses,

assuming the same mPy surface coverage. The data show that

REF decreases with spacer thickness, supporting the view that

the surface enhancement generated at the Ag surface is mainly

responsible for the observed SER intensity. Nevertheless, even

for a 10 nm thick spacer, REF has decreased only by a factor of

ca. 5 compared to bare Ag. The decrease in REF as a function

of SiO2thickness may be considered to be linear with a slope of

Δlog(REF)/Δd= 0.06 nm−1.

3.4. SERR Spectroscopy of Cyt c at Pt. Intense SERR

(surface-enhanced resonance Raman) spectra are obtained for

the heme protein cytochrome c (Cyt c) attached to SER-active

electrodes under violet light excitation which matches the

electronic transition of the heme chromophore. Furthermore,

the redox properties of Cyt c are preserved if the metal

electrode is coated by biocompatible self-assembled monolayer

(SAM). Cyt c is therefore widely used as a reference system to

test surface enhancement and electrical communication of

novel nanostructured electrodes.

21,35

Cyt c directly adsorbed on bare Ag surfaces is known to

undergo a partial transition to a non-native high-spin state of

the heme which can be distinguished from its native state in the

SERR spectrum.

36,37

To avoid this adsorption-induced protein

denaturation the electrodes are usually coated with a SAM of

ω-carboxyl alkanethiols. The increased biocompatibility,

however, is achieved at the expense of surface enhancement

that drops by a factor of 2 after coating with SAMs of

mercaptoundecanoic acid (MUA). For Cyt c directly adsorbed

to the Pt surface in Ag−SiO2−Pt electrodes SERR spectra with

high intensity could be obtained. However, the spectra also

indicated a strong contribution of the non-native high-spin state

(Figure S2 Supporting Information) accompanied by a time-

dependent decrease in SERR intensity. The Pt surface was

therefore coated with MUA prior to Cyt c adsorption, in

analogy to previous experiments on Ag surfaces.

The

electrodes were incubated in a 0.2 μM Cyt c containing

aqueous solution for 30 min, rinsed subsequently with

abundant buffer solution, and finally mounted into a

spectroelectrochemical cell containing a protein-free buffer

solution. Spectra obtained this way were stable over time and

could be quantitatively described by a superposition of spectra

of the native ferrous and ferric form of Cyt c only. The SERR

spectra of Cyt c attached to Ag−MUA and Ag−S−Pt−MUA

with S = MPTES−APTES differ only slightly in SERR

intensity, which is lowered by roughly a factor of 2 for the Pt

hybrid system (Figure 3A). By changing the applied potential of

the spectroelectrochemical cell the protein could be completely

reduced and reoxidized when adsorbed on the Pt surface, which

demonstrates that the electrical communication between the

working electrode and Cyt c is still intact. The Cyt c midpoint

potential was determined to be Em= 20 mV, which is identical

to its value on Ag−MUA.

REF on the Pt surface was obtained by comparison of the

relative Cyt c SERR intensities attached to Ag−MUA and Ag−

S−Pt−MUA supports. Thus, the REF on Ag−MUA was

estimated to be 2.5 ×102taking into account the effect of the

MUA coating on surface enhancement (vide supra). In order to

eliminate the effect on signal intensity that arises from

variations in the number of probed Cyt c molecules CV

measurements were carried out concomitant to the spectro-

scopic investigations. CV plots of Cyt c attached to either Ag−

MUA or Ag−S−Pt−MUA electrodes are shown in Figure 3B.

Integration of the voltammetric peaks allowed one to determine

the Cyt c surface coverage ΓCyt for both systems. ΓCyt was

calculated for Ag−MUA to be 6.5 ×10−11 Mcm

−2with respect

to the geometrical area of the electrode. On Ag−S−Pt−MUA a

nearly 2 times higher value of 1.2 ×10−10 Mcm

−2was

determined. One has to note that these values represent

Figure 2. (A) From bottom to top: SER spectra of mPy attached to Ag

(black), Ag−MPTS−APTES (light gray), and Ag−MPTS−APTES−Pt

(gray) surfaces. (B) Raman enhancement factors (REF) of mPy

attached to Pt as a function of the spacer thickness. Value marked with

an asterisk corresponds to mPy on bare Ag surfaces system.

Figure 3. (A) Potential-dependent SERR spectra of Cyt c on (from

top to bottom) Ag−MUA (E= +150 mV), Ag−S−Pt−MUA (E=

+150 mV), Ag−S−Pt−MUA (E=−400 mV), Ag−MUA (E=−400

mV). (B) CV of Cyt c on Ag−MUA (black) and Ag−S−Pt−MUA

(gray). S= MPTES−APTES. Scan rate: 100 mV/s.

Langmuir Article

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apparent surface concentrations as the respective surface

roughnessofthedifferentelectrodesisnotconsidered.

Multiplication of ΓCyt with the area of the laser spot gives the

number of Cyt c molecules that contribute to the observed

Raman signal. Thus, REF on Pt was corrected for the higher

number of probed Cyt c molecules and is presented in Figure

4A as a function of SiO2spacer thickness. In Figure 4A also the

REF values previously determined for Ag−SiO2−Au−MUA

electrodes

21,39

are shown. As can be seen, the overall value for

REF will be slightly higher if one uses Au as the outer metal.

Nevertheless, the slope of a linear fit in Figure 4A, which was

determined to be Δlog(REF)/Δd= 0.025 nm−1for Pt coatings,

is nearly the same for Au and Pt hybrid electrodes.

3.5. Calculations. The electric field enhancement distribu-

tion was calculated using the finite element method for a

electrode model geometry. The multilayered electrode depicted

in Figure 5A was modeled as follows: Starting with an infinite

long bulk Ag electrode (ydirection) with a height of 100 nm, 3

half spheres were attached with a radius r0= 42.5 nm

corresponding to the experimentally determined average Ag

coral size. The distance between the half spheres is unevenly

distributed with two spheres being in close vicinity (6 nm) and

a third one in a wider distance (62.5 nm). This structure is

coated with a 2 nm dielectric spacer of SiO2and a 5 nm thin Pt

film of the same surface morphology. Water was taken as the

surrounding medium. The applied external electromagnetic

field is taken as a plane wave incoming from the right,

propagating along the xdirection. The light is assumed to be

linearly polarized parallel to the yaxis, the wavelength is set to λ

= 413 nm, and the vector amplitude is set to |E



0|= 1. For the

chosen geometry it can be clearly seen that surface enhance-

ment of the fields is still present at the Pt−H2O interface. Due

to the choice |E



0|= 1 the field enhancement is given by the

absolute value of the electric field |E



|.Maximumfield

enhancement is achieved in the interspaces of the two close-

by half spheres. The perfect coated uniform Pt film in Figure

5A (2D plot left side), however, does not resemble the Pt film

that is present in the experiments. One of the main differences

compared to the real samples lies in the fact that the Pt island

film, as shown in Figure 1, is not completely covering the

underlying electrode, i.e., it exhibits hole-like defects. To

approximate the experimental conditions more closely, holes

were introduced into the Pt film shown in Figure 5B.

Interestingly, these defects enhance the average electric field

at the Pt surface. Figure 5 (right side) shows the corresponding

3D plots of the field enhancement for the perfect and

disordered system. It can be seen that introducing hole-like

defects generates more “hot spots”for surface enhancement in

the SiO2/Pt interface. Most remarkable are the “hot spots”at

the top of the half sphere and in the wider gap between the two

spheres where no surface enhancement was observed in the

absence of defects. It has to be further noted that a particularly

large enhancement is observed at the sharp edges of the Pt

islands, which are missing in closed films.

To obtain the average SERS enhancement for molecules

adsorbed on Pt, |E



|was read out at equidistant points 1 nm

above the Pt surface for the geometries shown in Figure 5A and

5B, respectively. The SERS enhancement of adsorbates placed

at these positions is then given by g=|E



|4.

Direct comparison

of both types of Pt films shows that the average SERS

enhancement per surface area is increased by approximately

80% for the disordered film.

4. DISCUSSION

High-quality SER(R) signals in Ag−SiO2−Pt constructs could

be seen for nonresonant Raman (mPy) and RR probes (Cyt c),

suggesting that the optical properties of the adsorbate itself do

not play a role in the magnitude of induced surface

enhancement at the Pt surface. Depending on the thickness

of the spacer layer the surface enhancement at the Pt layer was

estimated to reach 15−50% of the enhancement of pure Ag.

This is 2 orders of magnitude higher than the intrinsic

enhancement of pure Pt, which roughly reaches 0.2% of the Ag

value.

Furthermore, the comparable SERR enhancement

achieved for Pt and Au films in similar hybrid systems indicates

that the chemical nature of the outer metal island film is also

not highly relevant for the magnitude of induced SER activity.

These results suggest that the in situ Raman detection method,

proposed in this work, most likely can be applied for a variety

of metal films and adsorbates.

The surface enhancement of rough Ag electrodes was

determined to be 4 times higher at 514 nm compared to 413

nm laser excitation. The same wavelength-dependent enhance-

ment ratio was observed on Pt if thin spacer layers (1.7 nm)

would be used. This finding supports our hypothesis that the

optical properties solely of Ag are responsible for the observed

enhancement at the outer metal film. However, the distance-

dependent decrease of the REF was 3 times stronger for 514

nm excitation than for 413 nm. It might be that different spots

or local areas in the coral-like structures of Ag and Pt are

responsible for field enhancement at 514 and 413 nm. For

thinner coatings the wavelength-dependent field enhancement

distribution is not altered, whereas thicker coatings that have a

stronger influence on the overall surface morphology shift the

enhancement distribution to lower wavelengths.

This raises the question whether geometrical parameters of

the multilayer hybrid system in general are the main factor for

the magnitude of SER/SERR enhancement at the Pt surface.

The morphology of the Pt island film (Figure 1) shows a

coral-like nanostructure with smaller dimensions than the corals

of the underlying Ag. Furthermore, the Ag−SiO2substrate

contains areas with no or nondetectable Pt coverage. Both

effects were simulated in the field enhancement calculations

shown in Figure 5 by introducing small holes into the Pt film.

Figure 4. Raman enhancement factors (REF) for Cyt c on Ag−SiO2−

Au−MUA (open squares, taken from ref 32) and Ag−SiO2−Pt−MUA

(solid squares, this work) electrodes as a function of SiO2spacer

thickness.

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As a result, the average SERS enhancement is increased by ca.

80%.

This finding can be rationalized on the basis of two factors.

First, the presence of holes will increase the number of incident

photons on the Ag surface for excitation of surface plasmons

and thus for field enhancement, whereas for closed Pt film

losses due to reflection are much more severe. Second, the

defects also create sharp edges within the Pt nanostructure.

According to theoretical predictions, such a geometric

anisotropy in nanostructures provides a non-negligible

contribution to the overall SER enhancement.

41,42

This so-

called lightening rod effect focuses electric fields at the tip of

metallic ellipsoids constituting an additional contribution to the

field enhancement generated by metal particle plasmons. For

Pt, which displays only a poor field enhancement due to

particle plasmon resonances, such geometrical aspects are

considered as the most important parameter for its marginal

intrinsic SER activity.

In view of these considerations, we conclude that the

morphology of the Pt film plays the most crucial role for its

observed high SER activity. Simple electrochemical deposition,

as proposed in this work, creates Pt islands on top of coated Ag

nanostructures with a favorable geometry for induced surface

enhancement. This hypothesis is further supported by SERR

measurements on Ag−SiO2−Au hybrid electrodes where the

outer Au film was formed via sputtering and thus exhibited a

largely defect-free surface morphology. For this multilayer

system no SERR signals of Cyt c could be detected.

5. CONCLUSION

This work presents a detailed investigation on how SER

spectroscopy can be optimized in thin metal films that do not

have intrinsic plasmonic activity. In the present Ag−spacer−Pt

devices the SER intensity reaches ca. 50% of its value for Ag

under otherwise identical conditions. The separation of Pt from

Ag by a dielectric spacer has only a small effect on the surface

enhancement, but the silica coating efficiently prevents

Figure 5. Field enhancement calculations of Ag−SiO2−Pt geometries using a defect-free (A) and defect-containing (B) Pt film. On the right side the

corresponding 3D plots of the field enhancement are displayed.

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interaction of Ag with the adsorbates. As the most crucial

parameter for the magnitude of SER enhancement the surface

morphology of the metal island film could be identified,

whereas the chemical nature of the metal seems to play only a

minor role. The defect-containing outer Pt layer allows an

efficient excitation of the Ag plasmons that are required to

enhance the electric field in the vicinity of the Pt surface. The

anisotropic shape of the Pt corals additionally promotes surface

enhancement via the lightning rod effect. In summary, our

results suggest that geometrical parameters of such multilayered

electrode dominate the induced SERS efficiency on intrinsically

SER-inactive metal films. Hence, the present approach should

be applicable for analysis of adsorbates also on other types of

metals and thus may contribute to establish SER spectroscopy

as a versatile in situ analytical tool for various applications.

■ASSOCIATED CONTENT

SSupporting Information

Further electrode characterization by SEM and SERRS; SERR

spectra of Cyt c directly on Pt. This material is available free of

charge via the Internet at http://pubs.acs.org.

■AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Notes

The authors declare no competing financial interest.

■ACKNOWLEDGMENTS

The authors thank Peter Hildebrandt for encouragement and

support. Furthermore, we thank Ralph Krahnert and Ulrich

Gernert for their support in SEM measurements and the

Konrad-Zuse-Zentrum Berlin for providing the Maxwell solver

JCMsuite (www.jcmwave.com). Financial support from the

Fonds der Chemie and the DFG (Unicat, SPP Nanooptics) is

gratefully acknowledged.

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Danksagung

Ich m¨

ochte mich bei allen bedanken, die mich w¨

ahrend dieser Zeit unterst¨

utzt haben. Mein beson-

derer Dank gilt dabei folgenden Personen:

•Prof. Dr. Peter Hildebrandt f¨

ur die Betreuung w¨

ahrend der Doktorarbeit und f¨

ur seine

Unterst¨

utzung in jeglicher Hinsicht.

•Dr. Inez Weidinger f¨

ur die Betreuung und die fruchtbare Zusammenarbeit.

•Dr. Diego Millo f¨

ur die Zusammenarbeit, die vielen netten Gespr¨

ache und die Initiierung der

unvergesslichen "Hilde-Band".

•Dr. Anna Fischer f¨

ur anregende Unterhaltungen und die SEM-Messungen.

•Prof. Dr. Uwe Schr¨

oder, Dr. Falk Harnisch und Siang-Fu Hong f¨

ur die Bereitstellung der

Geobacter-Proben.

•Prof. Dr. Miguel A. de la Rosa und Dr. Irene Diaz-Moreno f¨

ur die nitrierten Cytochrom-

Proben.

•Prof. Dr. Andreas Knorr und Diplom Physiker Christoph K¨

ohler f¨

ur die Feldberechnungen.

•Dr. Uwe Kuhlmann f¨

ur Seine Hilfe im Umgang mit den Spektrometern.

•Claudia Schulz f¨

ur Ihre Hilfe im Labor.

•J¨

urgen Krauss und Dr. Hendrik Naumann f¨

ur Ihre Hilfe bei technischen Problemen.

•Lars Paasche f¨

ur Seine stete Hilfsbereitschaft bei allen Fragen.

•Meinen Freunden und B¨

urokollegen Jacek Kozuch und Francisco Velazquez-Escobar f¨

ur die

nette Arbeitsatmosph¨

are und die gemeinsame Zeit.

•Meinem alten Weggef¨

ahrten Murat Sezer mit dem ich so manche Stunden im Labor verbracht

habe.

•Marius Horch, Tillmann Utesch und allen weiteren Stammg¨

asten des Mittagstisches. Der

sch¨

onste Freitag bleibt unvergesslich.

•Marina B¨

ottcher, Sara Bruun, D¨

orte DiFiore, Dr. Jiu-Ju Feng, Nina Heidary, Julia Hellmich,

Anke Keidel, Dr. Friedhelm Lendzian, Wiebke Meister, Nobert Michael, Dr. Maria Andrea

Mroginski, Yvonne Rippers, Johannes Salewski, Dr. Eberhard Schlodder, Gal Schkolnik, Elis-

abeth Siebert, Dr. Arumugam Sivanesan, Dr. Neslihan Tavraz, Dr. Ingo Zebger und allen weit-

eren Mitgliedern des Max-Volmer Laboratoriums f¨

ur eine angenehme Arbeitsatmosph¨

are.

•Nele Bensmann f¨

ur Ihre Unterst¨

utzung.

Meiner lieben Familie!

123

Selbst¨

andigkeitserkl¨

arung

Die selbstst¨

andige und eigenh¨

andige Anfertigung dieser Arbeit versichere ich an Eides statt.

_______________________

Hoang Khoa Ly

Berlin, 15.08.2012

125