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Spectroscopic Evidence for Reversible Disassembly of the [FeFe] Hydrogenase Active Site Patricia Rodríguez-Maciá, Edward J. Reijerse, Wolfgang Lubitz, James A. Birrell, and Olaf Rüdiger J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b01608 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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The Journal of Physical Chemistry Letters

Spectroscopic Evidence for Reversible Disassembly of the [FeFe] Hydrogenase Active Site Patricia Rodríguez-Maciá, Edward Reijerse, Wolfgang Lubitz, James A. Birrell* and Olaf Rüdiger*

Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34–36, 45470, Mülheim an der Ruhr (Germany)

AUTHOR INFORMATION

Corresponding Author

E-mail: [email protected], [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: [FeFe] hydrogenases are extremely active and efficient H2-converting biocatalysts. Their active site comprises a unique [2Fe] sub-cluster bonded to a canonical [4Fe-4S] cluster. The [2Fe] sub-site can be introduced into hydrogenases lacking an assembled H-cluster through incubation with a synthesized [2Fe]H precursor, which initially produces the COinhibited state of the enzyme. Here, we present FTIR spectro-electrochemical studies on the COinhibited state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, DdHydAB. At very negative potentials disassembly of the H-cluster and dissociation of the [2Fe] sub-cluster is observed. Subsequently raising the potential allows co-factor rebinding and H-cluster reassembly. This demonstrates how the stability of the [2Fe]-[4Fe-4S] inter-cluster bond depends on the applied potential and the presence of an inhibiting CO ligand on the [2Fe] sub-cluster. These results provide insight into the mechanisms of CO inhibition and H-cluster assembly in [FeFe] hydrogenases. A fundamental understanding of these properties will provide clues for designing better H2-converting catalysts.

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KEYWORDS FTIR spectroscopy, electrochemistry, inorganic cofactor, hydrogen, carbon monoxide

Hydrogenases are the catalysts used by nature to reversibly convert H2 into H+ and electrons. They do this extremely efficiently; achieving high rates at minimal overpotentials.1-2 Learning how these highly optimized enzymes function may provide clues to develop better bio-inspired molecular catalysts. Hydrogenases can be divided into three groups ([NiFe], [FeFe] and [Fe] hydrogenases) depending on the metal content of their active sites.1 The [FeFe] hydrogenases are particularly active but they are also very sensitive towards molecular O2.3-5 In this group of hydrogenases catalysis occurs at a specialized cofactor called the H-cluster, which consists of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently linked through a conserved cysteine to a unique [2Fe] sub-cluster ([2Fe]H). [2Fe]H is composed of two iron ions bridged by an azadithiolate (adt) ligand, a bridging CO ligand and two terminal CN- and CO ligands (one on each iron) (Figure 1). These ligands stabilize the binuclear iron core in low oxidation states (I and II). Furthermore, they allow the facile study of these enzymes by means of spectroscopic techniques such as FTIR. The two iron ions at the active site are called proximal (Fep) and distal (Fed) with respect to [4Fe-4S]H. The Fed features an open coordination site where substrates (H2 or H+) as well as inhibitors (e.g. CO or O2) can bind. Within the [FeFe] hydrogenases there are several sub-classes that contain accessory FeS clusters (the so called F-clusters). The F-clusters function as electron-transfer relays to electronically connect the buried H-cluster with the protein surface.6-7



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Figure 1. Structure of the CO-inhibited form of the H-cluster of DdHydAB also showing the Fclusters. The intrinsic CO and CN- ligands are colored green/red and green/blue, respectively, and the exogenous CO is colored bright blue. The cysteine bridging the two parts of the H-cluster Cys382 is indicated. The figure is based on PDB: 1HFE.6

For [FeFe] hydrogenases, the H-cluster is assembled in two stages in vivo: the canonical [4Fe4S] cluster is inserted by the standard iron-sulfur cluster (ISC) system8 and the [2Fe]H sub-cluster is synthesized by the maturases HydE and HydG9, and finally inserted by a third maturase HydF.10-11 Inactive “apo” [FeFe] hydrogenases, containing [4Fe-4S]H but lacking the [2Fe] subcluster can be produced recombinantly in E. coli and the H-cluster can be reconstituted using synthetic [2Fe] cofactors, resulting in fully active “holo” enzymes when using [2Fe]adt as a cofactor.12-14 Armstrong and co-workers have studied artificial H-cluster assembly in CrHydA1 4 ACS Paragon Plus Environment

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from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum using protein film electrochemistry. Their results showed that artificial H-cluster assembly proceeds in a number of steps.15 Assembly begins with tight binding of the [2Fe]adt complex to the inactive “apo”hydrogenase, followed by attachment of [2Fe]adt to [4Fe-4S]H via a thiolate ligand. The latter step was found to be potential dependent suggesting that the redox state of [4Fe-4S]H controls this process. The final step, the release of a CO molecule to give the open co-ordination site on Fed proceeds rapidly yielding the catalytically active enzyme.16 Although this process is complex it occurs spontaneously. Understanding how the protein scaffold controls this process may allow the development of similar scaffolds to produce H-cluster analogs, through such simple and spontaneous chemistry.

During the catalytic cycle, the two Fe ions as well as the [4Fe-4S] cluster alter their oxidation states.1 The cycle is quite well understood for the F-cluster-free [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii, CrHydA1.1,

17-18

Five different active states of the

enzyme have been spectroscopically characterized: Hox, Hred HredH+, HsredH+ and Hhyd.19-23 The Hox state can bind exogenous CO forming the Hox-CO state.24,25 For CrHydA1, one-electron reduction of the Hox-CO state yields the Hred-CO state with a reduced [4Fe-4S]H (‫ܧ‬ு೚ೣ ି஼ை/ுೝ೐೏ ି஼ை = -470 mV, vs SHE at pH 8).25 Further reduction causes CO release forming the HsredH+ state (‫ܧ‬ுೝ೐೏ ି஼ை/ுೞೝ೐೏ ு శ = -500 mV, vs SHE at pH 8.0) (Figure 2).25



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Figure 2. Scheme describing the events taking place during reduction of CO-inhibited CrHydA1 hydrogenase.25 All potentials are quoted versus the standard hydrogen electrode (SHE).

In contrast to these findings, EPR experiments with the [FeFe] hydrogenase from Clostridium pasteurianum (CpI) indicated that CO binding at very negative potential can cause irreversible damage to the H-cluster.26 Similarly, protein film electrochemistry experiments demonstrated that CO inhibition is not completely reversible at negative potentials (during H+ reduction) while it is fully reversible at positive potentials (during H2 oxidation).16 This effect has been investigated in detail by Léger and co-workers using protein film electrochemistry in combination with density functional theory calculations.27 Their calculations suggest that CO bound to the Hred(H+) state (which they proposed to have a homovalent Fe(I)Fe(I) configuration at [2Fe]H) causes cleavage of the bond between Fep and the bridging cysteine, in disagreement with the observation by FTIR spectro-electrochemistry with CrHydA1that further reduction of the Hred-CO state releases CO and forms the HsredH+ state.25

Here, we study the heterodimeric [FeFe] hydrogenase from Desulfovibrio desulfuricans (DdHydAB) inhibited with CO by FTIR spectro-electrochemistry. Among all [FeFe] hydrogenases, DdHydAB is one of the most active bidirectional [FeFe] hydrogenases studied so


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far. Therefore, it is important to investigate this enzyme in detail to learn how nature has tuned its properties to achieve such high efficiency, and find inspiration for making synthetic molecular catalysts similarly effective. DdHydAB contains two [4Fe-4S] clusters (F-clusters) in addition to the H-cluster. These clusters appear to influence the properties of the H-cluster, lowering the potential at which the HsredH+ state is observed.24 Such an effect may also influence the stability of the reduced, CO-inhibited forms of the enzyme. Furthermore, DdHydAB is known to be much more strongly inhibited by CO than CrHydA1.16 For both these reasons we expect to observe a different behavior for the reduced, CO-inhibited form of DdHydAB at very negative potentials than was observed for CrHydA1, reflecting differences in the active site properties between different [FeFe] hydrogenases.

Figure 3A shows the spectra recorded in a spectro-electrochemical titration experiment at several different applied potentials and titration curves for the different species are shown in Figure 3B and C. In this experiment, the sample is transferred into a transmission FTIR cell containing a standard three electrode electrochemical system. The redox states of the enzyme are titrated as the applied potential is stepped under equilibrium/steady state conditions. This process is monitored by measuring how the current through the working electrode changes as the potential is equilibrated (Figure S1). The IR bands of the CO and CN- ligands are used to follow the appearance/disappearance of each redox species.1 From the titration curves obtained (Figure 3B and C), the midpoint potentials of the respective redox states can by calculated by fitting the data to the Nernst equation. At less negative potentials (-280 mV), a spectrum corresponding to the previously described Hox-CO state can be observed (Figure 3A, Spectrum i).24,25 As the potential is stepped to more negative values (-600 mV), a mixture of the Hox-CO and Hred-CO states is 7 ACS Paragon Plus Environment


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obtained (Figure 3A, Spectrum ii). The IR bands for the Hred-CO state are essentially the same as those described previously for CrHydA1.25 In the Hred-CO spectrum all the bands are shifted by 3-18 cm-1 to lower wavenumbers with respect to Hox-CO characteristic for reduction at [4Fe4S]H. These small shifts are due to a slight increase in the electron density on [2Fe]H, which weakens the CO and CN bonds and lowers νCO and νCN. Reduction on [2Fe]H leads to much larger shifts of around 50 cm-1.25 The much lower signal intensity observed at -600 mV is partially due to lower extinction coefficients of the IR bands for the Hred-CO state as compared to those for the Hox-CO state.25 At even more negative potential (-670 mV, Spectrum iii), the contribution from the Hred-CO state increases relative to Hox-CO, but it is clear that both states are decreasing in intensity compared with the spectra at more positive potentials. Spectrum iv (-740 mV) shows a complete loss of both the Hox-CO and Hred-CO states, and appearance of broad IR bands. This spectrum is essentially identical to that obtained when the [2Fe]adt complex is measured outside the protein in the same buffer (Spectrum v). This indicates that at very negative potentials the CO-bound cofactor dissociates from the active site.


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Figure 3. A: FTIR spectra obtained from the spectro-electrochemical redox titration of COinhibited DdHydAB in 50 mM MES, 50 mM HEPES at pH 8 and 150 mM KCl (as supporting electrolyte) containing redox mediators (see experimental section and Supplementary Discussion for details). The potential is stepped from less negative (a value close to the open circuit potential of -280 mV) to more negative (reductive titration) and subsequently back to less negative (oxidative titration), and spectra are measured at each potential. Selected spectra are shown to highlight the Hox-CO (light blue) and Hred-CO (green) states and free [2Fe]adt (pink). B: reductive titration curve, representing the intensities of the main peaks in each state (2017 cm-1 and 2002 cm-1) plotted against the potential. The solid line corresponds to the fits to the Nernst equation, from which the midpoint potential (E) and the number of electrons (n) can be derived. C: the subsequent oxidative titration curve. All potentials are quoted versus the standard hydrogen 9 ACS Paragon Plus Environment


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electrode (SHE). The unassigned peaks in A are attributed to the unavoidable incorporation of small amounts of degraded [2Fe]adt complex (probably the monocyanide form) during the artificial maturation process.14 Chronoamperometric measurements for the titration are shown in Figure S1.

In Figure 3, plot B and C show the titration of the normalized FTIR absorbance of the main bands in the Hox-CO and Hred-CO states (2017 cm-1 and 2002 cm-1) against the applied potential for the reductive and oxidative titrations, respectively. The solid line represents the fitting of the data using the Nernst equation. Fitting of the reductive titration gives midpoint potential values of -570 mV for the Hox-CO/Hred-CO transition and -700 mV for the loss of the Hred-CO state. The Hox-CO/Hred-CO transition in DdHydAB is 100 mV more negative than in CrHydA1.25 The potential of the HredH+/HsredH+ transition is also more negative in DdHydAB than in CrHydA1, a phenomenon attributed to the F-clusters.21,24 Some non-Nernstian behavior can be observed for the Hox-CO curve (light blue trace) in the region between -510 to -570 mV, which may also be related to the F-clusters. For the Hred-CO curve (green trace), the n-value of ≈2 at E0 = -700mV, derived from the fitting to the Nernst equation, does not indicate reduction by two electrons but instead the reduction of the Hred-CO state is coupled to a chemical step, namely the dissociation of [2Fe]adt, on a faster time-scale than the experiment, resulting in an apparent n=2 value.

As the potential is raised to less negative values, the characteristic sharp bands from the maturated protein reappear, as observed during de novo maturation of inactive “apo” DdHydAB.28 This suggests re-maturation of the enzyme is occurring as the available free [2Fe] co-factor rebinds. In contrast to the reductive titration, in the oxidative wave the Hred-CO state was not observed. Instead the Hox-CO state immediately appeared upon re-insertion of [2Fe]adt. 10 ACS Paragon Plus Environment

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This suggests that oxidation of the [4Fe-4S]H is required before re-insertion of [2Fe]adt, in good agreement with the potential dependence observed for the artificial maturation of CrHydA1.15 Because maturation of DdHydAB is a very slow process28 the re-insertion of the cofactor occurs on a time-scale slower than the potential scan rate, resulting in a lower apparent number of electrons in the titration. The Nernst fitting of the oxidative titration gives an apparent midpoint potential for [4Fe-4S]H reoxidation of ≈ -400 mV. It should be noted that the Hox-CO signal intensity only recovered to ≈5% of the original signal intensity. This low recovery may be due to the lack of stability of the [2Fe]adt in water and at low redox potentials for the long time periods used for this experiment, i.e. the sample spent 14 h at < -600 mV. An excess of [2Fe]adt is used during maturation of the protein to compensate for the complex degradation during the long maturation of inactive “apo” DdHydAB.28 In the redox titration a stoichiometric amount of [2Fe]adt is released from the active site. During the course of the experiment part of this released [2Fe]adt degrades and as a result, the recovery is only partial. When the experiment is titrated to just -700 mV, before all the Hred-CO signal intensity is lost (Figure S2), 70% of the initial signal intensity is recovered. In this experiment the sample spent just 5 h at < -600 mV.

We decided to investigate this effect in more detail by following the loss of signal intensity at very negative potentials with time. The data shown in Figure 4 correspond to an experiment in which the potential was poised at fixed values and spectra were measured at time intervals. Plot A shows the changes in the FTIR intensity of the main peaks from the Hox-CO (2017 cm-1, light blue circles) and Hred-CO states (2002 cm-1, green circles) as well as [2Fe]adt (1942 cm-1, pink circles) over time at different applied potentials. Plot B represents the potential steps over time applied during the experiment. The experiment was initiated at open circuit potential and immediately (t = 0) switched to -580 mV. This caused the Hox-CO signal intensity to decrease 11 ACS Paragon Plus Environment


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and the Hred-CO signal intensity to increase over the first 30 min, after which the signal intensities are stable. When the potential is switched to -680 mV, the bands from the Hox-CO state disappear (light blue) and the bands from the Hred-CO state (green) increase in intensity during the first 10 min. This is followed by the slow decrease in the Hred-CO state, which coincides with the appearance of the broad bands in the spectrum from the “free” [2Fe]adt. When the potential is stepped incrementally to even more negative values the rate of decay in the signal intensity of Hred-CO remains fairly constant, suggesting that the electron transfer is not rate limiting but the cofactor release. Returning the potential to -580 mV again does not recover the Hred-CO state. Instead, only when the potential is increased to more positive values, where the Hox-CO state originally dominated, do the Hox-CO state bands re-appear. This seems to occur quickly in the first 100 minutes and more slowly thereafter, reaching constant signal intensity after 24 h. In this experiment around 50% of the signal is recovered compared with the 5% recovered in the first experiments. This shows that shorter time periods at very negative potentials prevent degradation of the free complex.


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Figure 4. A: changes observed in the FTIR absorbance of the main peaks in the Hox-CO (light blue) and Hred-CO (green) states (2017 and 2002 cm-1 respectively) and [2Fe]adt (1942 cm-1) over time at different applied potentials during spectro-electrochemistry. B: the potential steps applied during the experiment. The experiment started at open circuit potential (around -280 mV) and was immediately shifted to -580 mV, followed by subsequent steps to more negative potential, then back to -580 mV and finally to -420 mV. The experiment was performed on a CO-inhibited DdHydAB sample in 50 mM MES, 50 mM HEPES at pH 8 and 150 mM KCl (as supporting electrolyte) mixed with redox mediators (see experimental section). All potentials are quoted versus the standard hydrogen electrode (SHE). The associated chronoamperometric measurements are shown in Figure S3.



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The behavior observed here for CO-inhibited DdHydAB is in stark contrast to that observed in CrHydA1. The latter undergoes a reduction event at -500 mV causing CO release and formation of the HsredH+ state.25 In DdHydAB, the [2Fe]adt complex dissociates preferentially. Our results are in line with those of Lèger and co-workers27 who, although lacking spectroscopic data, proposed that [2Fe]H dissociation occurs in an Hred-CO state with a homovalent Fe(I)Fe(I) configuration. We observe an Hred-CO state with the same properties as that observed in CrHydA125, i.e. a mixed valence [2Fe]H sub-cluster. Further reduction to an Hsred-CO state is likely to occur at [2Fe]H giving the homovalent Fe(I)Fe(I) species. Due to the large excess of electron density, this state is highly unstable and forces the release of either the exogenous CO or the complete [2Fe]adt sub-cluster (Figure 5). The reason why DdHydAB undergoes Fep-S-Cys bond cleavage rather than Fe-CO bond cleavage may be related to the much higher affinity of DdHydAB towards CO compared to other [FeFe] hydrogenases.16 Recently, Stripp and coworkers have suggested that the Hox-CO state is more stable with an apical CN ligand than an apical CO ligand on Fed and that ligand rotation is required for CO dissociation.29 Thus if the protein surrounding in DdHydAB increases the kinetic barrier for ligand rotation compared with CrHydA1, this could explain the slower CO dissociation in DdHydAB. This inactivation process is essentially the reverse of H-cluster maturation and, as we demonstrate here, its potential dependence agrees well with the results of Armstrong and co-workers for H-cluster maturation.15


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Figure 5. Scheme showing the proposed mechanism of the interconversion of the various redox states of the H-cluster observed for [FeFe] hydrogenases in the presence of CO. We predict reduction of Hred-CO gives a highly unstable state due to the large amount of electron density on [2Fe]H and immediately decomposes by release of either the exogenous CO, as observed for CrHydA1 (a. ref25), or [2Fe]adt, as observed here for DdHydAB. The bond between the cysteine and [2Fe]adt is only formed when the [4Fe-4S]H is oxidized. Subsequent to the Fep-S-Cys bond formation, the binuclear site is oxidized to Fe(I)Fe(II) by the oxidized [4Fe-4S] cluster (b. ref15), which requires the transfer of one extra electron to the F-clusters forming the Hox-CO state. The exogenously bound CO is colored in cyan. All potentials are quoted versus the standard hydrogen electrode (SHE).

We have demonstrated that the processes of CO-inhibition and H-cluster maturation are closely related. Maturation, i.e. formation of the bridging Fep-S-Cys bond can only occur at an oxidized [4Fe-4S]H cluster, which immediately oxidizes the CO inhibited [2Fe]H sub-cluster to the mixed



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valence Fe(I)Fe(II) state: Hred-CO. This state is stable in both CrHydA1 and DdHydAB but the rate of CO release could depend on the protein matrix. Further reduction of Hred-CO either leads to CO release (as in CrHydA125) or [2Fe]H detachment, i.e. “reverse maturation” (as we observe here in DdHydAB), explaining why under very reducing conditions CO inhibition is only partially reversible. The potential-dependent reversible disassembly of the H-cluster in the COinhibited enzyme reported here provides important insight into the mechanisms of CO-binding and co-factor assembly in the [FeFe] hydrogenases.

EXPERIMENTAL SECTION

DdHydAB was recombinantly expressed in E. coli as an inactive “apo” hydrogenase and artificially maturated following the procedure described in ref. 28. Active “holo” hydrogenase, DdHydAB, was inhibited with CO by flushing the sample with 100% CO gas for 30 min. Subsequently, the protein sample was mixed with the redox mediators anthraquinone-1,5disulfonic acid (Em7 = -234 mV, vs SHE), anthraquinone-2-sulfonate (Em7= -277 mV, vs SHE), benzyl viologen (Em= -358 mV, vs SHE), methyl viologen (Em= -449 mV, vs SHE) and 1,1',2,2'tetramethyl-[4,4'-bipyridine]-1,1'-diium iodide (Em= -540 mV, vs SHE) to a final concentration of 0.89 mM of CO-inhibited DdHydAB and 0.5 mM of each of the five redox mediators. The utility and stability of these redox mediators have been previously demonstrated.17 The mediators do not contribute to the IR spectra in the region of interest. Finally the sample was transferred into a spectro-electrochemical cell containing a semi-transparent gold mesh as the working electrode. DdHydAB is stable when in contact with the gold mesh, probably due to the transient nature of the interactions between the enzyme and the gold mesh. A piece of platinum was used as a counter electrode. An Ag/AgCl electrode was connected to the spectro-electrochemical cell 16 ACS Paragon Plus Environment

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and used as a reference electrode. All potentials are quoted versus the standard hydrogen electrode (SHE). The FTIR spectro-electrochemical setup consists of a homebuilt electrochemical cell, constructed according to the original design by Moss and co-workers.1, 30 The FTIR spectroelectrochemical cell assembly is described in detail in a tutorial from Gutierrez-Sanz et al..31 A Bruker IFS 66v/S FTIR spectrometer equipped with a nitrogen-cooled Bruker mercury cadmium telluride (MCT) detector was used to record the FTIR spectra in the double-sided, forward-backward mode, with an aperture setting of 1.5 mm, a scan velocity of 20 Hz and 1 cm-1 resolution. The sample temperature was maintained at 10 oC. The reference electrode was calibrated before and after each measurement using FcMeOH as described previously,32 to ensure that the potential of the reference electrode was stable during the course of the experiment. The potential was controlled by an Autolab PGSTAT101 potentiostat using Nova software with an equilibration time of 24 min at each potential until the current through the cell was stable, followed by continuous application of the potential during the measurement of the spectra. Data were processed using home-written routines in the MATLABTM environment. The Supporting Information includes a discussion about general remarks on FTIR spectroelectrochemistry (Supplementary Discussion).

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Supplementary Discussion – General remarks on FTIR spectro-electrochemistry Figure S1 - Chronoamperometry during the spectro-electrochemical redox titration. 17 ACS Paragon Plus Environment


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Figure S2 - FTIR spectro-electrochemistry from -300 mV to -700 mV Figure S3 - Chronoamperometry during spectro-electrochemistry.

AUTHOR INFORMATION

The authors declare no competing financial interests.

ACKNOWLEDGMENT

The

authors

would

like

to

thank

Ingeborg

Heise

for

the

synthesis

of

(Et4N)2[Fe2(adt)(CO)4(CN)2]; Patricia Malkowski and Nina Breuer help with the apo-enzyme preparation, as well as Birgit Nöring for her technical assistance in the electrochemical measurements, and Krzysztof Pawlak for assistance with FTIR measurements. This study was funded by the Max Planck Society and by the Cluster of Excellence RESOLV (EXC1069) from the Deutsche Forschungsgemeinschaft (DFG).


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[FeFe] Hydrogenase Active Site - ACS Publications - American

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