2
nd
coordination sphere controlled electron
transfer of iron hangman complexes on electrodes
probed by surface enhanced vibrational
spectroscopy†
H. K. Ly,*
a
P. Wrzolek,
b
N. Heidary,
a
R. G¨
otz,
a
M. Horch,
a
J. Kozuch,
a
M. Schwalbe*
b
and I. M. Weidinger*
a
Iron hangman complexes exhibit improved catalytic properties regarding O
2
and H
2
O
2
reduction, which are
attributed to the presence of a proton donating group in defined vicinity of the catalytic metal centre.
Surface enhanced resonance Raman (SERR) and IR (SEIRA) spectro-electrochemistry has been applied
concomitantly for the first time to analyse such iron hangman porphyrin complexes attached to
electrodes in aqueous solution. While the SERR spectra yield information about the redox state of the
central iron, the SEIRA spectra show protonation and deprotonation events of the 2
nd
coordination
sphere. To investigate the influence of a proton active hanging group on the heterogeneous electron
transfer between the iron porphyrin and the electrode, two hangman complexes with either an acid or
ester functional group were compared. Using time resolved SERR spectroscopy the electron transfer
rates of both complexes were determined. Complexes with an acid group showed a slow electron
transfer rate at neutral pH that increased significantly at pH 4, while complexes with an ester group
exhibited a much faster, but pH independent rate. SEIRA measurements were able to determine directly
for the first time a pK
a
value of 3.4 of a carboxylic hanging group in the immobilized state that shifted to
5.2 in D
2
Obuffer solution. The kinetic data showed an increase of the heterogeneous electron transfer
rate with the protonation degree of the acid groups. From these results, we propose a PCET which is
strongly modulated by the protonation state of the acid hanging group via hydrogen bond interactions.
Introduction
Second coordination sphere assisted reactions are crucial for
the efficiency of numerous catalytic transformations. For
example, in nature a variety of different reactions are catalysed
by heme cofactors where selectivity of the reaction is induced by
acidic or basic amino acids in the coordination sphere around
the heme environment.
1–3
This highly ordered arrangement of
proton donating or accepting groups denes the catalysed
reaction by the heme group, which can range from reduction of
hydrogen peroxide to water (catalase), substrate oxidation
(peroxidase), binding of molecular oxygen (myoglobin, hemo-
globin), oxygen reduction to water (cytochrome c oxidase) or
hydroxylation of different compounds (cytochrome P450).
Synthetic biomimetic molecular catalysts copy the essence of
the reaction centres of their enzymatic analogues, and exploit
the optimally evolved active structures for maximal perfor-
mance.
4,5
In this respect, they exhibit numerous advantages
compared to their biological idols. On the one hand, due to
their smaller size, better substrate accessibility and higher
stability, these compounds bear a high potential to be used in
technological applications such as biomimetic fuel cells. On the
other hand, the study of molecular catalysts is highly valuable in
general. Well-dened synthetic catalysts allow detailed investi-
gations of the catalytic mechanism at a molecular level and
precise ne-tuning of desired catalytic activity using synthetic
chemistry. Understanding the structure–function relationships
of catalytically active sites can in turn enhance the knowledge of
biological catalysis.
One of the challenges in catalyst design is to mimic the
electron/proton transfer interplay that has been naturally opti-
mized in enzymatic catalysis. In this regard, hangman
porphyrin complexes that carry a heme group and an arbitrary
functional “hanging”group positioned in a dened distance to
the reaction centre, constitute an interesting model system to
study the inuence of the 2
nd
coordination sphere.
6–8
Hangman
a
Department of Chemistry, Technische Universit¨
at Berlin, PC14, Straße des 17. Juni
135, D-10623 Berlin, Germany. E-mail: inez.weidinger@tu-berlin.de; khoaly@
mailbox.tu-berlin.de
b
Department of Chemistry, Humboldt Universit¨
at zu Berlin, Brook-Taylor-Str. 2, D-
12489 Berlin, Germany. E-mail: matthias.schwalbe@hu-berlin.de
†Electronic supplementary information (ESI) available: Details on data treatment
procedure for TR-SERR and SEIRA spectroscopy and electrocatalysis. See DOI:
10.1039/c5sc02560e
Cite this: Chem. Sci.,2015,6, 6999
Received 15th July 2015
Accepted 4th September 2015
DOI: 10.1039/c5sc02560e
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complexes that exhibit a hanging carboxylic acid group have
been shown to signicantly enhance the catalase
9
and oxidase
reaction in solution in comparison to complexes with non-
acidic hanging groups.
10,11
For similar iron hangman corroles, a
catalase like reaction mechanism has been proposed that
involves the carboxylic acid group as a proton donor site.
12
Cobalt hangman porphyrins and corroles have also been
successfully tested in electrocatalytic dioxygen reduction and
hydrogen evolution.
13–15
For a technological application in fuel
cells, the hangman complexes have to be immobilized on an
electrode surface. In contrast to homogeneous reactions, the
adsorption provides numerous advantages such as site isolation
of catalytically active centres, facilitated catalyst recycling and
the general use of aqueous solvents.
16–19
Importantly, the
created direct electronic contact can lead to enhanced electron
transfer (ET) between catalyst and electrode.
20
ET processes play
a crucial role in the electrocatalytic mechanism as reaction
intermediates are generated through electron acceptance/
donation. Therefore, the rate of this process may not only
determine overall catalytic activity but has also been shown to
directly inuence the reaction products in case of oxygen
reduction.
21
The study of adsorbed compounds is challenging and
requires adaptation of suitable spectroscopic methods that
provide structural insights into the catalytic processes at the
surface. The elucidation of these heterogeneous reactions is a
major prerequisite for promoting technological application of
hangman compounds. In this regard, surface enhanced vibra-
tional spectroscopy has the surface sensitivity to investigate
sub-monolayer concentrations of immobilized molecules. In
particular, the two vibrational spectroscopic techniques,
surface enhanced Raman (SER) spectroscopy and surface
enhanced infrared absorption (SEIRA), are able to provide
different and oen complementary information at a molecular
level that can be used to monitor both, redox changes and
protonation events of adsorbed compounds. Particularly, for
heme containing molecules, laser excitation with violet light
allows exploitation of the molecular resonance effect yielding
surface enhanced resonance Raman (SERR) spectroscopy to
selectively monitor the vibrational modes of the absorbing
porphyrin ring. Hence, SERR spectro-electrochemistry has been
used extensively in the past, in particular, to analyse the redox
and catalytic properties of surface bound heme enzymes.
22–26
Recently, also SERR measurements of surface bound heme
containing molecular catalysts were presented providing inter-
esting insights into their catalytic mechanism by inter alia
monitoring direct product transformation at the heme using a
RDE-SERR setup.
27–32
SEIRA spectroscopy on the other hand
monitors all vibrations of the surface bound molecules. It is,
however, especially sensitive to polar vibrations, such as
carboxylic acid groups, and has been used in the past e.g. to
analyse the protonation of a single glutamic acid residue in a
complex protein matrix.
33
The combination of both types of
surface enhanced vibrational spectroscopies has been applied
to understand the effect of protein reorientation in enzymatic
electrocatalysis.
34
In the present work it is used for the rst time
to study small electrocatalytic active complexes on surfaces and
to correlate electron transfer with proton delivery events in the
coordination sphere. Thus, this technique is able to provide
unique insight into the 2
nd
coordination sphere controlled
heterogeneous electron transfer (HET) of molecular catalysts on
surfaces in operando. In this paper, we present the rst results
regarding electron and proton transfer processes of surface
bound heme based hangman complexes in the absence of
substrate using SERR and SEIRA spectroscopy.
Materials and methods
Iron hangman porphyrin compounds were synthesised
according to published procedures.
6,11
Briey, the free base
porphyrins POH and POMe were synthesised as described in
ref. 6 and reacted with iron(II) chloride in dimethylformamide
as described in ref. 11. Aerobic acid workup yields in the
formation of the corresponding chloroiron(III) porphyrin
complexes.
11
For SERR measurements, an electrochemically roughened
Ag ring electrode was used as solid support prepared by a
previously described procedure.
35
For SEIRA measurements, a Si
prism was coated chemically with a thin Au layer that was used
as electrode interface. A detailed description of the process and
measurement geometry can be found here.
36
The respective
electrodes were incubated overnight (>16 h) in an ethanolic
solution containing 0.6 mM and 0.3 mM of 1-heptanethiol
(98%, Sigma Aldrich) and 1-(11-mercaptoundecyl)imidazole
(96%, Sigma Aldrich), respectively. This procedure leads to the
formation of a mixed self-assembled monolayer (SAM) on top of
the electrode's surface. The electrodes were cleaned with
abundant ethanol prior to use. Hangman adsorption was ach-
ieved by incubation of the SAM coated electrodes with a ca. 10
mM solution of the hangman compound in DCM.
28
Immobili-
sation was nished aer 2 h, and unspecic bound, i.e. phys-
isorbed, compounds removed by rinsing with abundant DCM
(>99.8%, Sigma Aldrich).
28
The electrodes were subsequently mounted into a home-
made spectro-electrochemical cell prepared for potential
controlled SERR experiments and rotated (10 Hz) during
measurements to avoid laser induced degradation. Rotation of
the electrode is further necessary to minimize diffusion limi-
tation of substrate or protons.
37
SEIRA measurements were
carried out using a home-built spectro-electrochemical cell in
the ATR mode in Kretschmann geometry using the Si prism as
waveguide.
36
For measurements in aqueous phosphate buffer
(PBS) solution, an Ag/AgCl 3 M KCl reference electrode was used
(DriRef, WPI). Unless otherwise mentioned, PBS buffer always
refers to pH 7 and 100 mM concentration. Catalysis tests were
performed using diluted H
2
O
2
(30% in water, Sigma Aldrich) in
buffer using a commercial rotating Au disc electrode setup (Pine
Instruments). All employed solvents and chemicals were
purchased and used without further purication. All experi-
ments were performed under Ar atmosphere.
SERR spectra were acquired using the 413 nm line of a
krypton ion laser (Coherent Innova 300c) coupled to confocal
Raman setup with a single-stage spectrograph (Jobin Yvon
LabRam 800 HR) equipped with a liquid-nitrogen-cooled CCD
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detector in 180back scattering geometry. The laser light was
focused using a Nikon 20objective (N. A. 0.35) with a working
distance of 20 mm. Laser power on the sample was about 1 mW.
Spectra acquisition times varied from 5 to 60 s for stationary
and from 120 to 180 s for time resolved measurements,
respectively. All experiments were repeated several times to
ensure reproducibility. For time resolved (TR) SERR experi-
ments, potential jumps of variable height and duration were
applied to trigger the redox reaction as previously described.
38
The SERR spectra were measured at different delay times
following the potential jump using synchronized laser light
modulators. Aer background subtraction the spectra were
treated by component analysis, in which the spectra of indi-
vidual species, i.e. components, were tted to the measured
spectra using a home-made analysis soware.
39
SEIRA
measurements were carried out using a Bruker IFS 66v/s spec-
trometer equipped with a photoconductive MCT detector. 400
scans were co-added for a spectrum with a nal resolution of
about 4 cm
1
.
Results and discussion
Two iron hangman porphyrin complexes with a different
hanging functional group were synthesized according to pub-
lished procedures.
6,11
In the rst complex, the hanging group
consists of a proton active carboxylic acid terminus while the
second exhibits a carboxylic ester group (see Fig. 1). The former
complex is abbreviated as FePOH, the latter as FePOMe.
Immobilization of the hangman complexes on SERR active Ag
supports was achieved by coating the supports with a mixed
monolayer following a recently published procedure.
28
The
monolayer consists of two types of molecules: a shorter methyl
terminated (HS–(CH
2
)
6
–CH
3
) and a longer imidazole termi-
nated (HS–(CH
2
)
10
–Im) alkanethiol. Specic binding of the
hangman compound is expected to occur by coordination of the
imidazole nitrogen to the heme iron as present in heme–
histidine systems of biological heme enzymes. Unspecic
bound compounds were removed by rinsing with abundant
dichloromethane (DCM). Subsequently, the solution was either
changed to acetonitrile (ACN) or PBS buffer. As a rst step, it
was checked whether the hangman complexes could preserve
their catalase function upon immobilization. For this, catalytic
catalase activity of the immobilized hangman complexes
towards H
2
O
2
oxidation was detected by chronoamperometry. A
rotating Au electrode coated with SAM and hangman complexes
was immersed into a 100 mM PBS pH 7 solution and the
potential set to +0.1 V. Upon stepwise H
2
O
2
substrate addition,
increasing catalytic currents were observed conrming catalytic
activity in the immobilized state in aqueous PBS buffer solution
(for details see ESI Section 1†). For the concentration of H
2
O
2
at
half maximum current, a value of 8 4 mM and 3 1mMH
2
O
2
was determined for FePOH and FePOMe, respectively. More-
over, a ca. threefold higher catalytic current was observed for
FePOH than for FePOMe under identical experimental condi-
tions (ESI Fig. S1†).
SERR spectroscopy of immobilised hangman complexes
In a second step, SERR spectroscopy was performed on the
hangman/electrode system using a rotating Ag ring electrode.
Intense SERR spectra of the immobilised hangman compounds
were obtained upon 413 nm Soret laser excitation. SERR spectra
recorded in the absence of the imidazole terminated alka-
nethiol linker molecule afforded no SERR signals (ESI Fig. S2†)
Fig. 1 (A) Structure of FePOH and FePOMe hangman complexes. (B)
Schematic representation of the hangman immobilization on elec-
trodes using a mixed imidazole terminated SAM.
Fig. 2 SERR spectra of FePOH in 100 mM PBS buffer at 0.4 V and
+0.15 V vs. Ag/AgCl 3 M with the respective component spectra: HS
red (red), HS ox (grey), LS red (green), LS ox (blue).
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supporting the proposed direct binding of the heme iron to the
imidazole (see ESI section 2†for more information). Fig. 2
shows the SERR spectra of the imidazole immobilised FePOH
hangman complex on Ag electrodes at 0.4 V and 0.15 V applied
electrode potential in Ar purged PBS buffer. The spectra
resemble typical spectra of heme compounds, exhibiting strong
marker bands around 1370 cm
1
(n
4
) and 1575 cm
1
(n
2
) as well
as a broader band with lower intensity at around 1495–1500
cm
1
(n
3
). These bands are indicative for the redox, spin and
ligation state of respective heme compounds.
40
To extract the
contribution of the different redox and congurational species,
a component t analysis was performed (for details of the tting
procedure see section 3 in ESI†).
39
Briey, known component
spectra of different heme types were used and modied
accordingly. As a result, the presence of two heme spin states
(high spin (HS) and low spin (LS)) were found, each appearing
in two different redox states (Fe
III
and Fe
II
). The molecular
nature of the different (spin) species is not known a priori.
However, regarding the surface functionality of the SAM, we
propose that the HS species is represented by an iron complex
with the imidazole group of the SAM as h axial ligand. A
water or hydroxide molecule loosely attached to the heme iron
as a sixth ligand is very likely and usually does not lead to a
change in spin state (vide infra).
40
To induce the observed LS
state, a stronger binding sixth ligand is required. The nature of
this 6
th
ligand is yet unknown and it might be a residue of the
synthesis procedure or an unwanted side-product that is
formed on the electrode surface. This species furthermore
shows only a limited redox activity (vide infra). For the following
data evaluation, we therefore concentrate on the Fe–HS species.
To transform SERR intensities into relative surface concen-
trations, spectral intensities of the different heme species,
determined from the component analysis, were multiplied with
respective SERR cross sections accounting for the different RR
scattering efficiency and summed up to a total intensity.
40–42
Relative concentration of a particular heme species was derived
by calculating the relative spectral intensity of this species in the
overall intensity. Calculation and determination of the cross
sections followed established procedures for heme proteins (for
details see section 4 in the ESI†).
40–42
The relative surface
concentrations of each species are shown in Fig. 3A as a func-
tion of applied potential. At the starting potential of 0.15 V, a
mixture of oxidized HS and LS species is observed with the HS
species as the major fraction. A reduced species that contains
both, a LS and a HS conformation, arises at more negative
potentials at the expense of the oxidized HS species. In contrast,
the concentration of the oxidized LS species seems to be largely
independent from the applied electrode potential.
At of the Nernst equation to the values of the oxidized HS
species as a function of potential yields the redox potential E
0
for the redox couple Fe
III
/Fe
II
–HS. The so derived values for E
0
of
FePOH and FePOMe are plotted in Fig. 3B as a function of pH.
Here, signicant differences are observed for the two types of
hangman complexes. While FePOH shows a distinct depen-
dence of E
0
on pH, E
0
of FePOMe remains almost pH inde-
pendent. A linear t of the data for FePOH yields a slope of 57
5mVpH
1
.
SEIRA spectroscopic probing of the hanging group
In contrast to SERR measurements, SEIRA experiments can
visualize non-heme-related changes. For SEIRA measurements,
the hangman complexes were immobilized in the same way as
in the SERR experiment albeit in this case a nanostructured
gold lm deposited on a Si prism was used as electrode.
36,43
First, adsorption of the SAM onto the electrode was followed.
Here, a band pattern in the region from 3000 cm
1
to 2800 cm
1
and a band at 1113 cm
1
is observed (Fig. S3†). These bands can
be attributed to modes with high contributions from C–H
stretching vibration due to the methylene groups of the SAM
molecules.
44
Furthermore, a band at 1510 cm
1
is observed,
which is assigned by comparison with literature and by DFT
calculations to the n(C]C) stretching mode of the deprotonated
imidazole ring.
45
A more detailed discussion on the band
assignment is provided in section 2 of the ESI.†Fig. 4 shows the
SEIRA spectrum of the immobilized FePOH and FePOMe
compound in ACN solution using the SAM coated electrode as a
Fig. 3 (A) Relative contributions of the FePOH component spectra
(Fe
III
–HS: open triangles, Fe
II
–HS: solid triangles, Fe
III
–LS: solid
squares, Fe
II
–HS: open squares) as a function of electrode potential.
(B) Redox potentials of FePOH (squares) and FePOMe (circles) as a
function of pH.
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reference. Upon addition of the compounds, the imidazole
band at 1510 cm
1
disappears or shis conrming that the
hangman complexes indeed bind via the proposed coordinative
Fe/N(imidazole) bond. Furthermore, for FePOH a prominent
band arose at around 1737 cm
1
that can be assigned to the
n(C]O) stretching vibration of the protonated carboxylic acid of
the hangman motif. In the case of FePOMe, this band is located
at around 1727 cm
1
in accordance with an expected downshi
for carbonyl stretching frequencies of esters with respect to
acids.
44
For FePOH and FePOMe, a shoulder at 1706 cm
1
is
observed, which is more pronounced for FePOMe. As this band
is observed for both complexes, we exclude that it originates
from the carbonyl stretching vibration of the hanging group
itself. More likely, the band may arise from a high shied n(C]
N) vibration probably of the heme pyrrole or the imidazole C]N
group. Interestingly, the n(C]O) vibration of FePOH dis-
appeared when the solution was changed to aqueous PBS buffer
at pH 7. This observation can be explained with a deprotonation
of the carboxylic acid group. Upon changing the pH of the
buffer solution to low pH values, the band at 1737 cm
1
reap-
peared clearly associated with a decrease in intensity of the
band at 1565 cm
1
as shown in Fig. 5A. This band most likely
represents the asymmetric n(COO
) stretching of the associated
carboxylate base. The intensity of the 1737 cm
1
band was used
to create a pH titration curve presented in Fig. 5B. From these
measurements, we determine the pK
a
value of the hanging acid
group in aqueous solution to be 3.4 0.2. Upon D
2
O exchange,
the band of the FePOH shis to 1715 cm
1
(ESI Fig. S7†). This
constitutes a rather drastic downshiand might be caused by
an additional overall change in the hydrogen/deuteron bonding
network around the acid group. The pK
a
of the acid group in
D
2
Obuffer was determined to be 5.2 0.4 (Fig. 5B). Qualita-
tively such a shiin pK
a
is in line with a predicted increase of
basicity upon deuteration of carboxylic acids.
46
Finally, SEIRA
difference spectra were measured as a function of potential in
ACN (with 10% MeOH) and PBS buffer. In both cases, no
potential induced changes of the 1737 cm
1
band were
observed (ESI Fig. S8†) indicating that the protonated/depro-
tonated form of the carboxylic hanging group is stable over the
scanned potential range.
TR-SERR spectroscopic determination of the HET rate
Using time resolved SERR spectroscopy, the heterogeneous
electron transfer rates k
HET
of FePOH and FePOMe in aqueous
and deuterated phosphate buffer solution were measured by
following the oxidation state of the heme as a function of time.
38
Measurements were performed at pH 7 and pH 4 to investigate
the inuence of the protonation state of the carboxylic acid
group on the ET kinetics. The relative contribution of the
Fig. 4 SEIRA spectrum of immobilized FePOH (black) and FePOMe
(blue) in ACN. The SAM coated Au electrode was used as reference
spectrum.
Fig. 5 SEIRA difference spectrum of FePOH in PBS buffer of different
pH. From bottom to top: pH ¼5.5, 4.3, 3.0 and 1.6. The spectrum at pH
7 was used as reference. (B) Normalized intensity of the 1737 cm
1
band (or the 1715 cm
1
band in D
2
O) as a function of pH.
Fig. 6 Relative contribution of the Fe
III
–HS (solid squares) and Fe
II
–HS
(hollow squares) species of FePOH as a function of delay time after a
potential jump from 0.15 V to 0.4 V.
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oxidized Fe
III
–HS species was monitored as a function of delay
time subsequent to a potential jump (see ESI section 6†). The
initial potential was set to 0.10 V. The nal potential was set in a
way to yield an overpotential of h¼EE
0
¼0.30 V taking into
account the difference in redox potential at different pH values.
For such a high overpotential, the rate observed for reduction
can be set equal to the heterogeneous electron transfer rate
k
HET
.
35
Fig. 6 shows a typical relaxation curve of the Fe
III
–HS
species to the surface redox equilibrium at the nal potential
induced by the potential jump. In all measurements, we
observed a fast initial drop of the Fe
III
–HS concentration that
was sometimes followed by a much slower relaxation phase
until equilibrium conditions were reached (ESI Fig. S11†). In
order to achieve a consistent evaluation of the kinetic data, all
kinetic traces were tted with monoexponential functions. The
rate constant is obtained by k
ET
¼1/swhere sis the time
constant of the t function (details on the TR SERR measure-
ments are given in section 6 of the ESI†).
35,38,41,42
The heteroge-
neous ET (HET) rate constants were obtained from several
different experiments and their respective mean values are lis-
ted in Table 1. Due to scattering of the data, an average relative
error of 15% for k
ET
is stated. The determined HET rates
obtained at signicantly high overpotential show values in the
order of several thousand per second. Among other factors,
these fast ET rates might be a result of the direct wiring of the
heme iron to the electrode affording a good electronic coupling
and/or electron tunneling path.
20
Again, a distinctly different
behavior of FePOH compared to FePOMe was observed. While
for FePOMe, the ET rates do not depend on the buffer pH within
the given accuracy, the ET rate of FePOH is more than 25 times
higher at pH 4 than at pH 7. A similar behavior is observed upon
switching to deuterated phosphate buffer solution. For
FePOMe, a slight decrease of the rate constant is observed upon
changing from pH 7 to pH 4 in D
2
O. In contrast, the rates
increased for FePOH by 6 times for the same set of measure-
ments. Comparing the rates at the same pH in H
2
Ovs. D
2
O,
another remarkable observation is made. FePOMe shows
almost no kinetic isotope effect (KIE) at both measured pH
values. In contrast, the ET rates of FePOH at pH 7 increase more
than tenfold affording, in fact, an inverse KIE ¼0.08. A smaller
inverse KIE ¼0.3 is observed at pH 4.
Mechanistic implications of the effect of the hanging group
The distinctly different behaviour of both compounds points to
a direct perturbation of the redox thermodynamic and kinetic
behaviour by the hanging group. While FePOMe shows almost
no variation of E
0
as a function of pH, FePOH exhibits a shiof
E
0
by 57 mV per pH unit. This nding strongly indicates a
PCET step involved in the redox transition of FePOH from Fe
III
/
Fe
II
. In this vein, the pH dependent shifurther implies a
transfer of one proton to the compound upon one electron
reduction.
31,47–49
A simple redox transition induced de-/proton-
ation of the carboxylic group was not observed in potential
dependent SEIRA experiments over a broad potential range and
can therefore not account as accompanied PT process. More
likely is the scenario of a water or hydroxyl ligand bound at the
heme iron as 6
th
ligand in the ferrous and ferric state, respec-
tively. This additional protonable ligand may be able to induce a
PCET reaction as found already for other transition metal (e.g.
Ru) complexes.
49,50
The existence of such a ligand is difficult to
probe spectroscopically as the addition of a hydroxyl or aqua
ligand does not change considerably the electronic congura-
tion of the heme. Therefore, RR spectra of ve coordinated and
six coordinated HS–heme complexes with an aqua/hydroxyl
ligand exhibit high resemblance with only minor alterations in
the low frequency region from 210–450 cm
1
.
51,52
Nevertheless,
hydroxyl as 6
th
ligand has been identied in the crystal structure
of the ferric FePOH and similar complexes have already been
reported for a range of other heme compounds.
7,51
Although OH
and H
2
Oas6
th
ligand exhibit, in general, only weak to moderate
binding affinities towards the heme iron, the presence of the
carboxylic acid hanging group might be able to stabilise the
ligation due to hydrogen bonding interactions.
51
In this sense,
the rigid carboxylic group xes the water/OH
molecule at the
heme iron cavity. A similar situation was already reported for a
picket-fence Fe–heme complex in which a Fe
II
–OH
2
was stabi-
lized by hydrogen bonding interaction with an amide group in
the 2
nd
coordination sphere.
51,52
Based on this, we propose a
Table 1 Electron transfer rates derived from TR SERRS in different
buffer and pH.
Buffer pH
k
HET
/s
1
FePOH FePOMe
PBS H
2
O 7 100 7000
PBS H
2
O 4 2800 7800
PBS D
2
O 7 1300 7700
PBS D
2
O 4 8600 5600
Scheme 1 Proposed reaction scheme for a proton coupled ET reac-
tion of the FePOH. The stated charge does not consider the hanging
group.
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reaction pathway of a possible PCET reaction of the FePOH
presented in Scheme 1. In this scheme, state 1,i.e. ferric FePOH,
carries a hydroxyl ligand that is protonated upon reduction to
the ferrous state. This means, the Fe
II
–OH
2
complex formation
is achieved through 1e
/1H
+
transfer. In this assumption, low
pH values would afford the thermodynamic destabilisation of
state 1in favour of state 2. This will lead to a facilitated
reduction, which is consistently perceived as a positive shiof
the redox potential upon lowering the pH. In the case of
FePOMe, the lack of hydrogen bond interactions might result in
a vacant axial position, thus, suspending a PCET reaction.
Alternatively, in line with the observations, is also a scenario of
ferric OH-bound FePOMe in which the hydroxyl ligand detaches
upon reduction. This may also explain the subtle increase of the
redox potential at lower pH, which affords lower hydroxide
concentrations in solution facilitating the detachment.
The kinetic data obtained for the HET between electrode and
the different hangman compounds supports the hypothesis.
Here, FePOMe shows almost no deviation of k
HET
upon
changing pH and isotopic exchange. This observation is in line
with both of the proposed scenarios for FePOMe above and
points to a fast and unimpeded direct ET process. Moreover, the
absolute rate constants lie in the range expected for direct
electrode-wired heme domains and most likely involves “pure”
electron tunnelling.
20
In contrast, a distinctly different behav-
iour is noted for FePOH as is expected for a PCET reaction.
53,54
Here, a drastic impact of the hanging group on the HET kinetics
is observed. Specically, a dependence of the HET rate on the
protonation degree of the hanging carboxylic acid group was
found. Fig. 7 shows the derived kinetic rate constants for FePOH
from Table 1 plotted against the protonation degree calculated
via the Henderson–Hasselbalch equation (see ESI section 7†):
1
10pHpKaþ1¼xCOOH (1)
x
COOH
denotes the molar fraction of the protonated carbox-
ylic acid group at a given pH value. A clear correlation between
HET rate and protonation degree can be seen in Fig. 7. The k
HET
rates could be tted reasonably with an exponential function.
The general dependence of the kinetic constants on the
protonation degree of the hanging group can be rationalised by
considering a perturbation of the ET/PT equilibrium. In this
regard, two major effects may have to be distinguished. In the
simplest view, FePOH carries either a protonated acid or
deprotonated carboxylate hanging function. These two different
pH dependent states exhibit a different net charge resulting in
an altered electrostatic environment close to the heme, and
altered hydrogen bonding interactions with the bound OH/OH
2
at the iron. Both factors are expected to exhibit a major impact
on the stability/energy of the different states 1–4. Therefore,
these factors might also signicantly modulate the pathway of
the PCET shown in Scheme 1. Following this argumentation,
the potential jump induced redox transition may also proceed
differently for the two species leading to the different observed
kinetic behaviour. Scheme 2 summarises the possible interac-
tions of the protonated and deprotonated acid with the 6
th
OH/
OH
2
ligand. Note that Scheme 2 is shown in a very minimalistic
way to highlight the different reaction pathways. In principle, it
cannot be excluded that an additional water molecule is placed
between the 6
th
ligand and the hanging group. This, however,
does not lead to a principle change in the proposed reaction
schemes. In the case of the protonated acid (Scheme 2A),
formed at lower pH values, a hydrogen bond interaction
between the acid function and bound OH is present that may
allow efficient formation of Fe
III
–OH
2
,i.e. state 2via PT1. Hence,
equilibrium between 1and 2is shied to the latter, and ET may
Fig. 7 Protonation degree of the carboxylic acid hanging group of
FePOH in % calculated via eqn (1) plotted vs. the TR-SERR spectro-
scopic derived k
HET
in Table 1. The dashed line represents an expo-
nential fit to the data.
Scheme 2 Proposed modulating interaction of the carboxylic acid
hanging function in the protonated (A) and deprotonated form (B) with
bound OH for FePOH. H
+
B
denotes a proton from the bulk. Indicated
charges refer only to the heme unit.
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predominantly proceed via state 2/4. In contrast, in the case
of the deprotonated acid function that lacks this H-bond, direct
ET1 from state 1/3is rather expected (Scheme 2B). PT2 would
then occur subsequent to ET by a proton from the bulk that
might be pre-coordinated at the carboxylate function (not
shown in the Scheme). As the TR-SERR spectroscopic experi-
ment, however, only follows changes in the heme redox state,
the water formation at the axial ligand binding site is not
monitored. Comparing the two ET routes, i.e. ET1 and ET2, one
would intuitively assume that latter is more efficient indepen-
dent from the protonation state of the acid group, affording
faster ET rates. In fact, ET1 involves a formation of the high
energetic intermediate 3that accommodates closely situated
negative charges. This is also in line with energetic consider-
ations that generally hold for PCET reactions.
54
In our system,
PT1 and ET1 are energetically uphill, while the corresponding
transfer reactions ET2 and PT2 are downhill.
49,53,54
Therefore,
one would expect an increase of ET rate constants upon
lowering the pH as ET2 becomes the dominating process.
Alternatively, the stepwise ET/PT reaction might also be
replaced by a concerted PCET reaction at neutral to basic pH
values to proceed directly from 1to 4circumventing the
formation of 3. The coupling of a fast ET to a most likely slower
PT process will afford signicantly decreased apparent HET rate
constants measured by TR-SERR spectroscopy, also in line with
our experimental observations.
54
Kinetic measurements in D
2
O
can reveal the existence of a concerted PCET as the isotopic
exchange would lead to a more pronounced deceleration of the
HET rate constants.
54
However, in our experiments the isotopic
exchange also afforded a distinctly shied pK
a
value of the
hanging group. The observed inverse KIE might therefore be
rather related to an acceleration of ET rates through shiof the
protonation/deuteration equilibrium in the same vein as
mentioned above. Furthermore, the possible existence of a
concerted PCET process, as proposed for these complexes
11,55
is
supported by the measurements of catalytic activity regarding
H
2
O
2
dismutation. As this reaction requires both electrons and
protons, its reaction rate will be controlled by the slowest of the
two charge transfer processes. At pH 7 catalytic activity of
FePOH is equal or even better than for FePOMe albeit the
apparent HET rate is 2 orders of magnitude lower. If a stepwise
ET/PT process would be present with a constant rate for PT, the
result should afford lower catalytic activity for FePOH at pH 7. In
a concerted PCET process, however, the slowest reaction could
be equally fast or even faster than in the case of FePOMe.
Although it is not possible to pin down unambiguously the
exact reaction route, we have conclusively shown that the
protonation of the hanging group is strongly inuencing the
HET of immobilised hangman complexes. This observation
points to a strong coupling of the HET rate with the availability
of protons in the 2
nd
coordination sphere. Interestingly, such
modulated ET has not been observed before in solution under
non-turnover conditions. However, homogeneous reactions
using an electrode as electron supplier afford slow ET rates
(10
2
cm s
1
).
15
It might very well be that the inuence of the 2
nd
coordination sphere becomes only observable when high HET
rates are present, which holds true for direct electrode wired
complexes.
20
This effect might be highly important for electro-
catalytic efficiency of surface bound hangman complexes, and
has to be investigated in the future in more detail.
Conclusions
For the rst time, the electron transfer properties of immobi-
lised iron hangman complexes were analysed in aqueous solu-
tion via surface enhanced vibrational spectroscopy. The
inuence of a proton active hanging group in the 2
nd
coordi-
nation sphere on the non-turnover redox thermodynamics and
kinetics of the hangman complexes was studied by investigating
two different hangman complexes that exhibit either an acid or
an ester functionality as hanging group. Signicant differences
were found for these two compounds as only the acid contain-
ing complex showed a strong dependence of redox potential
and heterogeneous ET kinetics on pH and H/D exchange.
Concomitantly performed SERR and SEIRA measurements
were able to correlate the HET rates to the pK
a
of the carboxylic
acid hanging group, which was determined experimentally for
the rst time in aqueous buffer solution. The obtained data
provides evidence for an increased HET rate with increased
protonation degree of the carboxylic acid function. As possible
explanation, a PCET reaction is proposed for the proton active
complex that is strongly modulated by the pH dependent redox
equilibrium of the hanging acid group. The overall ndings
shed light on the reaction mechanism of heterogenised
hangman complexes in aqueous environment and demonstrate
the impact of the 2
nd
coordination sphere on the redox and
kinetic properties of these catalysts immobilized on electrodes.
This effect might be of high relevance for the heterogeneous
catalytic activity of Fe hangman complexes or similar molecular
electrocatalysts. Finally, our research proves the capability of
the combination of (TR) SERR and SEIRA spectroscopy to probe
2
nd
coordination sphere mediated reactions.
Acknowledgements
The authors would like to thank Peter Hildebrandt, Uwe Ull-
mann, Lars Paasche and Ingo Zebger for valuable support;
Financial support from the DFG (excellence cluster “Unicat”
and CRC 1078 A1) is greatly acknowledged.
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