Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes [original]

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Metalloenzymes Hot Paper

Exploring Structure and Function of Redox

Intermediates in [NiFe]-Hydrogenases by an Advanced

Experimental Approach for Solvated, Lyophilized and

Crystallized Metalloenzymes

Christian Lorent,* Vladimir Pelmenschikov, Stefan Frielingsdorf,

Janna Schoknecht, Giorgio Caserta, Yoshitaka Yoda, Hongxin Wang,

Kenji Tamasaku, Oliver Lenz, Stephen P. Cramer,Marius Horch,*

Lars Lauterbach,* and Ingo Zebger*

In memory of Frank Koschine

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How to cite: Angew.Chem. Int. Ed. 2021,60,15854–15862

International Edition: doi.org/10.1002/anie.202100451

German Edition: doi.org/10.1002/ange.202100451

15854 T2021 TheAuthors.AngewandteChemieInternational Editionpublished by Wiley-VCHGmbH Angew.Chem. Int.Ed. 2021,60,15854–15862

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Abstract: To study metalloenzymes in detail, we developed

anew experimental setup allowing the controlled preparation

of catalytic intermediates for characterization by various

spectroscopic techniques.The in situ monitoring of redox

transitions by infrared spectroscopyinenzyme lyophilizate,

crystals,and solution during gas exchange in awide temper-

ature range can be accomplished as well. TwoO

2-tolerant

[NiFe]-hydrogenases were investigated as model systems.First,

we utilized our platform to prepare highly concentrated

hydrogenase lyophilizate in aparamagnetic state harboring

abridging hydride.This procedure proved beneficial for 57Fe

nuclear resonance vibrational spectroscopyand revealed, in

combination with density functional theory calculations,the

vibrational fingerprint of this catalytic intermediate.The same

in situ IR setup,combined with resonance Raman spectrosco-

py,provided detailed insights into the redoxchemistry of

enzyme crystals,underlining the general necessity to comple-

ment X-raycrystallographic data with spectroscopic analyses.

Introduction

Transition metals are often involved in chemical and

enzymatic catalysis.Innature,metal-containing enzymes

catalyze avariety of reactions,especially the conversion of

small gaseous molecules like CO2,N

2,orH

2.This type of

chemistry is relevant for establishing alternative strategies for

energy conversion and the production of carbon-neutral fuels.

Many of these metalloenzymes,including carbon monoxide

dehydrogenase,nitrogenase and hydrogenase,are attractive

targets for biotechnological application and can serve as

blueprints for bioinspired chemistry.[1–6] However,their

rational utilization requires athorough mechanistic under-

standing,typically requiring multiple spectroscopic tech-

niques.

Infrared (IR) spectroscopy in various implementations

has been successfully used to monitor catalytic processes and

intermediates at and beyond biological metal sites.Available

approaches comprise surface-sensitive techniques like sur-

face-enhanced infrared absorption (SEIRA) and/or attenu-

ated total reflection (ATR) spectroscopy,optionally com-

bined with electrochemistry,illumination, gas-atmosphere

and/or temperature control.[7–12] Recently,time-resolved IR

studies in the nanosecond range as well as ultrafast pump-

probe and two-dimensional IR techniques have also been

introduced into metalloenzyme research.[13,14] Electron para-

magnetic resonance (EPR) spectroscopy provides additional

structural and electronic insight into paramagnetic states,[15–18]

while resonance Raman (RR) spectroscopy has been success-

fully applied to monitor the characteristic metal@ligand

modes of specific redox states and cofactors.[19–24] Addition-

ally, 57Fe nuclear resonance vibrational spectroscopy (NRVS),

asynchrotron-based technique that can selectively probe

iron-specific normal modes,has been lately established for

characterizing iron-containing enzymes.[25–32]

Here,wehave designed anew experimental setup for

spectroscopic analyses of gas-converting metalloenzymes in

various sample forms.Gas composition and temperature can

be adjusted, thereby enabling the preparation of specific

redox states for subsequent characterization by complemen-

tary spectroscopic tools,such as RR, EPR, or NRVS.Addi-

tionally,redox transitions between catalytically relevant

intermediates and resting states can be monitored by in situ

IR spectroscopy.

To illustrate the versatility of our approach and its benefits

for exploring metalloenzyme catalysis,weinvestigated two

O2-tolerant model enzymes,the regulatory and the mem-

brane-bound [NiFe]-hydrogenase from Ralstonia eutropha

(ReRH and ReMBH, respectively). Briefly,[NiFe]-hydro-

genases catalyze the reversible cleavage of molecular hydro-

gen into protons and electrons (Figure 1). All of these

[*] C. Lorent, Dr.V.Pelmenschikov,Dr. S. Frielingsdorf,J.Schoknecht,

Dr.G.Caserta, Dr.O.Lenz, Dr.M.Horch, Dr.L.Lauterbach,

Dr.I.Zebger

Department of Chemistry,Technische Universit-tBerlin

Strasse des 17. Juni 135, 10623 Berlin (Germany)

E-mail:christian.lor[email protected]

[email protected]

[email protected]e

Dr.Y.Yoda

Japan Synchrotron Radiation Research Institute

SPring-8

1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198 (Japan)

Dr.H.Wang, Prof. Dr.S.P.Cramer

SETI Institute

189 Bernardo Avenue, Mountain View,California 94043 (USA)

Dr.K.Tamasaku

RIKEN SPring-8 center

1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

Dr.M.Horch

Department of Physics,Freie Universit-tBerlin

Arnimallee 14, 14195 Berlin (Germany)

E-mail:marius.horc[email protected]

Supportinginformation and the ORCID identification number(s) for

the author(s) of this article can be found under:

https://doi.org/10.1002/anie.202100451.

T2021 The Authors. Angewandte Chemie International Edition

published by Wiley-VCH GmbH. This is an open access article under

the terms of the Creative Commons Attribution License, which

permitsuse, distribution and reproduction in any medium, provided

the original work is properly cited.

Figure 1. Schematic representationofthe consensusstructure and

cofactor composition of the model [NiFe]-hydrogenases investigated in

this study.The redox state of the active site is primarily determined by

the oxidation state of the nickel ion and the nature of the bridging

ligand X.

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enzymes feature aheterodimeric core structure,consisting of

alarge subunit harboring the [NiFe] active site and asmall

subunit comprising iron–sulfur clusters that form an electron

transfer relay.The proposed catalytic cycle and redox

intermediates of [NiFe]-hydrogenases discussed in this study

are displayed in Figure S1.

Results and Discussion

Theexperimental setup introduced here is designed for

the preparation of well-defined redox states of metalloen-

zymes and their concomitant multi-spectroscopic analysis

under various experimental conditions and in different

sample forms.The sample compartment consists of agas-

tight chamber connected to an external vacuum pump and

avariety of dry or humidified gases (Figures 2and S2). The

temperature can be set between 80 Kand 333 K. Our newly

developed approach also allows the in situ analysis of protein

solutions (Figures S3 and S11), lyophilizates (Figure 3C)and

single crystals (Figures 5and S10) by IR transmission

spectroscopy.Since the whole sample compartment is por-

table and gas-tight, it can also be disconnected from the

remaining setup and moved (Figure S2), for example,for RR

spectroscopic analysis of the same sample or further sample

treatment in an anaerobic chamber.

To illustrate the capabilities of our platform, we first

describe the preparation and spectroscopic characterization

of highly concentrated lyophilized samples of ReRH, suitable

for conducting NRVS experiments.Upon incubation with H2,

ReRH resides predominantly in the so-called Nia-C catalytic

intermediate.This species harbors abridging hydride between

the FeII and NiIII ions (Figure S1), which is of general interest

in biological and chemical catalysis.[33–35] So far, experimental

evidence for the presence of the hydride in this state has only

been provided by EPR spectroscopy.[35–37] Currently,the only

vibrational spectroscopic method suitable to monitor metal@

ligand bonding in this light-sensitive species (Figure S4) is

NRVS.[19,20] Observation of active-site metal@ligand vibra-

tions with this technique,however,typically requires protein

concentrations above 1mm,[27,28] which is the upper concen-

tration limit for purified ReRH in solution. To overcome this

limitation, we used our setup for agentle lyophilization to

prepare highly concentrated enzyme samples while preserv-

ing their catalytic activity.NRVSwas performed subsequently

in combination with density functional theory (DFT) calcu-

lations to reveal structural details of the Nia-C state.

In order to obtain highly concentrated samples in the Nia-

Credox state,0.5 mmprotein solution of ReRH was flash-

frozen at 77 Kand transferred to the sample compartment

(Figures 2A and S2). Subsequently,water was removed at

controlled temperature and pressure by applying amild

vacuum of 0.1 mbar while slowly warming up the sample to

243 K. After completion of the lyophilization, the sample

compartment was flushed with dry H2gas to reduce the

enzyme.Finally,compression of the protein lyophilizate,

using aspatula and apestle,yielded highly dense samples

(equivalent to 4–5 mm,see Supporting Information for de-

tails) suitable for demanding methods such as NRVS (Fig-

ure 2C). Forfurther details,see the Materials and Methods

section in the Supporting Information and Figure S2.

To verify structural integrity and functionality of the

lyophilized oxidized and H2-reduced enzyme,wemeasured

the H2-oxidation activity and applied EPR, IR and RR

spectroscopy (Figure 3). This multi-spectroscopic approach

was only applicable due to the design of the setup.The IR

signature of the amide Iand amide II vibrational modes is

related to the absorption of the (polyamide) backbone of the

protein and therefore reflects its secondary and tertiary

structure.[38] Thesimilarity between the spectra of the

lyophilized and solution-phase samples indicates that the

freeze-drying process did not affect the integrity of the

protein structure (Figure 3A). Additionally,lyophilized and

Figure 2. Setup for in situ IR analysis of proteins. A) The sample

compartment consists of agas-tight, temperature-controlled chamber

with IR-transparent windows. Agas supply allows the exchange of

various dried or humidified gases (H2,N

2,O

2)and mixturesthereof.

B) Protein samples can be studied in multiple forms:insolution in

asandwich cell made of two IR windows (Figures 5, S3B and S11B),

as lyophilized powder (Figures 3Cand S3A) or in the crystalline phase

(Figures 5and S11A) on top of asingle window.C)The concentration

of aprotein sample can be significantly increased, up to an equivalent

of 4–5 mm,via lyophilization and subsequent compression.

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subsequently re-dissolved ReRH showed the same hydro-

genase activity as freshly isolated enzyme,indicating that the

metal cofactors and amino acid side chains responsible for

substrate conversion and proton/electron transfer were not

altered by lyophilization (Figure 3A inset). Theintegrity of

the iron–sulfur cluster relay was verified by almost identical

Fe@Ssignals in the corresponding RR spectra (Figure 3B).

Likewise,metal@ligand vibrations,characteristic for the most

oxidized, hydrogen-binding [NiFe] intermediate,termed Nia-

S(Figure S1), were observed, thereby confirming anative

active-site structure of oxidized ReRH lyophilizate (Fig-

ure 3B).[20]

Intriguingly,the lyophilized enzyme still reacts with

molecular hydrogen, as monitored by in situ IR spectroscopy

(Figure 3C). Thebiologically unusual CO and CN@ligands at

the active site of [NiFe]-hydrogenases give rise to valuable IR

marker bands that allow the identification of individual redox

states of the [NiFe] center.[39–41] TheH

2-dependent formation

of the Nia-C state indicates aredox-active ReRH in the

compressed and H2-reduced lyophilizate.Complementary

EPR spectra display the typical rhombic signature of the Nia-

Cstate,[35] confirming anative active-site structure after

lyophilization, reduction and compression (Figure 3D).

Thelyophilization procedure described above yielded

highly concentrated, 57Fe-enriched ReRH samples,which

Figure 3. Spectroscopic and biochemical characterization of lyophilized (lyo.) ReRH compared to freshly prepared enzyme solution (sol.). A) IR

spectroscopic signature reflecting the amide Iand II vibrational modes [38] of the oxidized enzyme. The inset shows the specific H2-oxidation

activity of freshly prepared, freeze-thawed and re-dissolved lyophilized samples. B) RR spectra of the oxidized enzyme obtained with 458 nm

excitationand normalizedtothe most intense active site signal at 550–552 cm@1.The small shifts of 1–2 cm@1likely reflect differences between

protein purified from cells grown with either 57Fe (lyo.) or iron with natural isotope distribution (sol.). The sharp signal marked with an asterisk

derives from an optical artefact of the Raman spectrometer.C)IRspectra of ReRH, oxidized (top), H2-reduced (middle) and subsequently re-

oxidized with air (bottom).For details about the assigned active-siteredox states, see Figure S1 and Table S1. D) EPR spectra of the H2-reduced

enzyme in solution and as compressed lyophilizate (both measured at 80 Kand with 1mWmicrowave power). IR and EPR spectra of the protein

solution are normalized to the intensity of the lyophilized sample.

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allowed NRVS-based detection of active-site vibrations of the

Nia-C state.The NRVS data shown in Figure 4comprise the

spectral regions characteristic for Fe@CO/CN modes (400–

650 cm@1)and Ni@H@Fe wagging vibrations (650–

800 cm@1).[25–28] Thecount rate of the elastic peak accumulated

by the detector increased by afactor of four for the

lyophilized sample in comparison to the solution-phase

sample (Figure 4A,inset), providing asignificant improve-

ment of the signal-to-noise ratio.Consequently,the error bars

of the two sample spectra vary,onaverage,byafactor of four

(Figure 4A,see Supporting Information for details).

In order to specifically probe the metal-bound hydride of

the Nia-C state,lyophilized ReRH samples were treated with

both H2and D2(Figure 4B). Theresulting experimental

spectra were compared to simulated data obtained by DFT

calculations (Figure 4B), based on an ReRH active-site

homology model[42,43] (Figures S5 and S6;for details,see the

DFT Methods section of the Supporting Information).

Analysis of the Nia-C spectrum obtained after incubation of

ReRH with H2(Figure 4B,top blue trace) revealed two

intense bands at 554 and 598 cm@1and aweaker one at

574 cm@1.Additional spectral features appear at 446, 470, 500

and 508 cm@1.Previously,[NiFe]-hydrogenase vibrational

signals at these energies were attributed to normal modes

dominated by Fe@CO and Fe@CN stretching and bending

motions.[19–21,25–28] Overall, the agreement between the exper-

imental and calculated spectra is good (Figure S7). The

isolated Ni@H@Fe wagging motions are predicted to produce

only very weak 57Fe-PVDOS intensities in the 670–740 cm@1

region. Themost noticeable spectral feature of this type is

expected at 694 cm@1and formed by two modes at 692 and

696 cm@1,each characterized by approximately 30% mH-

PVDOS but only subtle 1%57Fe-PVDOS.Adiscrete hydride

band could not be resolved by NRVS due to the low signal-to-

noise ratio in this spectral region, far away from the elastic

Mçssbauer peak. However,adistinct H/D-sensitive band

observed at 574 cm@1is predicted to arise from two modes

calculated at 574 and 576 cm@1(7% 57Fe-PVDOS each), both

of which are combinations of mHwagging and C@NH2

bending of the nearby Arg411 guanidium group (Figures S7A

and S8). TheNRVSdata recorded from ReRH incubated with

D2(Figure 4B,top red trace) reveal small shifts of 1–5 cm@1to

lower frequencies for several bands.This can be explained by

deuteride motions contributing to the Fe@CO(CN)-dominat-

ed modes,[20,44,45] with a mD-PVDOS maximum (23%) calcu-

lated at 501 cm@1for Nia-C(mD) (Figures S7B and S8, normal

mode animations provided in Supporting Information). Most

prominently,the 574 cm@1band detected for the H2-reduced

sample is absent in spectra from both the experimental D2-

treated sample and the calculated Nia-C(mD) model. More-

over, anewly emerging signal (calculated Nia-C(mD) mode at

581 cm@1)can be observed as aweak shoulder at ca. 586 cm@1,

close to the nearby high-intensity feature centered at

596 cm@1,indicating the presence of the mDdeuteride.In

summary,application of the novel setup proved to be

beneficial for the enrichment of ReRH, thereby enabling

NRVS characterization of the reduced enzyme.Rationalized

by DFT calculations,these data allowed insights into active-

site metal@ligand vibrations including normal modes that

reflect hydride coordination in the Nia-C redox state.

Using ReMBH as amodel metalloenzyme,wenext

demonstrate the applicability of our setup for the character-

ization of protein crystals by multiple spectroscopic tech-

niques (Figures 2B and S10). To obtain acomprehensive

understanding of redox processes in [NiFe]-hydrogenase

crystals,wefirst compared in situ IR spectra of aerobically

grown protein crystals and solution (Figure 5A,B). Unlike

Ash et al.,who employed electrochemical control in combi-

nation with redox mediators to induce redox transitions

within crystals of Hydrogenase 1from Escherichia coli,[46] our

design allows reduction of the hydrogenase with its native

substrate H2and (re)oxidation with the inhibitor O2.Thus,

our setup allows monitoring physiologically relevant redox

processes at the [NiFe] active site,asexemplified for the

Figure 4. Nuclear resonancevibrational spectra of ReRH in the Nia-C

state. A) NRVS data of lyophilized,H

2-reduced and compressed ReRH

(lyo.,black trace) compared to the corresponding protein solution at

0.5 mm(sol.,gray trace). Both spectra were recorded for 12 hours. The

inset displaysthe corresponding count rates at the elastic peak.

B) Experimental NRVS data of lyophilized ReRH (top) incubated with

H2(blue traces, 26 hours accumulation)orD

2(red traces, 16 hours

accumulation) in comparison to the corresponding DFT-calculated

57Fe-PVDOS spectra, which were obtained using the model shown in

Figure S6. NRVS data includingerror bars are displayed in Figure S9.

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