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His-ligation to the [4Fe-4S] sub-cluster tunes the catalytic bias of [FeFe] hydrogenase Patricia Rodríguez-Maciá, Leonie Kertess, Jan Burnik, James A. Birrell, Eckhard Hofmann, Wolfgang Lubitz, Thomas Happe, and Olaf Rüdiger J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11149 • Publication Date (Web): 14 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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Journal of the American Chemical Society

His-ligation to the [4Fe-4S] sub-cluster tunes the catalytic bias of [FeFe] hydrogenase Patricia Rodríguez-Maciá†#*, Leonie Kertess‡#, Jan Burnik∥, James A. Birrell†, Eckhard Hofmann∥, Wolfgang Lubitz†, Thomas Happe‡, Olaf Rüdiger†* Max Planck Institute for Chemical Energy Conversion, Stiftstraße 34-36, 45470 Mülheim an der Ruhr, Germany. Photobiology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44801 Bochum, Germany. ∥ Protein Crystallography, Faculty of Biology and Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44801 Bochum, Germany. † ‡

ABSTRACT: [FeFe] hydrogenases interconvert H2 into protons and electrons reversibly and efficiently. The active site H-cluster is composed of two sites: a unique [2Fe] sub-cluster ([2Fe]H) covalently linked via cysteine to a canonical [4Fe-4S] cluster ([4Fe4S]H). Both sites are redox active and electron transfer is proton-coupled, such that the potential of the H-cluster lies very close to the H2 thermodynamic potential, which confers the enzyme with the ability to operate quickly in both directions without energy losses. Here, one of the cysteines coordinating [4Fe-4S]H (Cys362) in the [FeFe] hydrogenase from the green algae Chlamydomonas reinhardtii (CrHydA1) was exchanged with histidine and the resulting C362H variant was shown to contain a [4Fe–4S] cluster with a more positive redox potential than the wild-type. The change in the [4Fe-4S] cluster potential resulted in a shift of the catalytic bias, diminishing the H2 production activity but giving significant higher H2 oxidation activity, albeit with a 200 mV overpotential requirement. These results highlight the importance of the [4Fe-4S] cluster as an electron injection site, modulating the redox potential and the catalytic properties of the H-cluster.

iron located further away from [4Fe-4S]H, the distal iron (Fed), harbors an open coordination site where substrates (H2 and/or H+) and inhibitors (such as CO and O2) bind.12

INTRODUCTION Hydrogenases are the H2 cycling catalysts in nature. The active sites of these enzymes contain earth abundant metals like Ni and/or Fe and they operate as efficiently as platinum.1 Thus, they represent a source of inspiration for the development of efficient H2 converting catalysts based on cheap and abundant metals.

It was recently shown that the apo-hydrogenase from Chlamydomonas reinhardtii HydA1 (which only contains the [4Fe-4S] cluster) could be reconstituted in vivo with synthetic model complexes in the presence13 and the absence of the HydF maturase.14 The artificial maturation of [FeFe] hydrogenases has permitted the study of many different variants of the H-cluster and especially, heterologous expression of the apo-hydrogenase in E.coli has greatly facilitated site-directed mutagenesis.15

Among the three main classes of hydrogenases ([NiFe] hydrogenases, [FeFe] hydrogenases and [Fe] hydrogenases),2 the [FeFe] hydrogenases are the most active (up to 10,000 s-1 in H+ reduction)3 and compared with the [NiFe] hydrogenases, they operate in both directions at very small overpotential even in the presence of H2.4 However, they are rapidly inactivated by trace amounts of O2, although this inactivation was shown to be partly reversible under certain conditions.5-9

The protein sequence surrounding the H-cluster is highly conserved in all hydrogenases characterized to date; in particular, the coordinating ligands of the [4Fe-4S]H cluster are invariably cysteine residues.10-11, 16 These residues coordinate iron by means of their side-chain thiolate group.17 However, histidine (His) serves as an alternative ligand for the surface-exposed [4Fe-4S] cluster of the [NiFe] hydrogenases and the [FeFe] hydrogenase form Clostridium pasteurianum,11 where the His-ligated clusters are thought to play an important role in electron transfer.18

The catalytic cofactor of the [FeFe] hydrogenase is the socalled H-cluster, which is formed from a unique [2Fe] subcluster ([2Fe]H) covalently linked to a canonical [4Fe-4S] cluster ([4Fe-4S]H) by a cysteine thiolate. In the binuclear [2Fe]H sub-cluster, the two irons ions are bridged through an azadithiolate (ADT) ligand. Additionally, both irons are coordinated by two terminal carbon monoxide (CO) and cyanide (CN-) ligands (one on each iron) and a bridging CO ligand.10-11 The iron directly bound to [4Fe-4S]H is called the proximal iron (Fep) and it is coordinately saturated, while the

The electron transfer sites in hydrogenases are an important target for protein engineering to control catalysis. However, the effects of non-Cys coordinating ligands on the redox and structural properties of iron-sulfur clusters remain not completely established for [FeFe] hydrogenases. Often site-

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were performed (Figure S2). The oxidized [4Fe-4S]2+ cluster is diamagnetic and, therefore, EPR silent, whereas the reduced [4Fe-4S]+ is paramagnetic and displays a typical axial S = ½ EPR signal around g = 2. The C362H variant appeared to contain 100% preformed [4Fe-4S] clusters, with no evidence for [3Fe-4S] clusters, which would give signals in the oxidized sample. The only signal present in the oxidized sample is a sharp isotropic signal from the thionine radical.

directed mutation of cluster ligands leads to incomplete cluster insertion or protein instability.19 In a previous work, we have demonstrated that the first coordination sphere of the [4Fe–4S] cluster in CrHydA1 can be altered using sitedirected mutagenesis by single exchange of each cysteine (C115, C170, C362, and C366) with various alternative amino acid residues such as alanine, aspartate, or serine. Only four of the variants (C170S/D and C362S/D) underwent correct [2Fe] cluster insertion. The catalytic bias and catalytic activity were influenced for these variants, yet all of them were found to be less active than the wild type enzyme.20

Unequivocal proof of a His-ligated [4Fe-4S] cluster was obtained by X-ray crystallography. Brown protein crystals grown under strictly anaerobic conditions could be obtained for the apo-HydA1 C362H variant (Figure S3). The crystal structure was determined at 1.80 Å resolution (PDB 6GL6) and confirms the replacement of the cysteine residue at position 362 by a histidine. In the variant, the inserted imidazole-nitrogen is positioned to coordinate the iron with a distance of 2.18 Å (Figure 1). This is within the range of NFe distances found in other His-ligated clusters (e.g. 2.16 Å in the His-ligated [4Fe-4S] cluster of the CpI [FeFe] hydrogenase27-28 and 2.15 Å in the His-[2Fe-2S] cluster of mitoNEET).29 All iron and sulfur atom positions of the [4Fe4S] cluster are fully occupied. An overall comparison of the crystal structure with the apo-HydA1 WT structure (PDB 3LX4)30 reveals an additional SO32- anion near the [4Fe-4S]H cluster, which derives from the presence of sodium dithionite in the protein solution. Possibly related, a shift of Thr171 and Arg172 as well as an alternative conformation of Cys170 are observed (Fig. S4 and S5). All residues as well as the SO32- anion are located at the protein surface allowing for a flexible orientation of the side-chains and subsequently for the observed shifts compared to the apo-HydA1 WT model.30 Overall, the structure of the C362H variant indicates conservation of the protein fold compared with the WT structure.

Recently, the catalytic cycle of [FeFe] hydrogenases has been intensively revisited and although there is no general consensus on the mechanistic details,21-23 proton-coupled electron transfer (PCET) between the two sub-clusters of the H-cluster is believed to be essential for efficient and reversible catalysis. However, the precise protonation site regulating the PCET at the H-cluster is still under debate.21, 24 It has been suggested that cysteine 362 is the protonation site of [4Fe-4S]H and that this protonation is coupled to the reduction of the [4Fe-4S] cluster, thereby affecting the catalytic cycle, i.e. the directionality and activity of the enzyme.25 In this regard, we decided to investigate the influence of a cysteine to histidine exchange at this position in the current work. This exchange is expected to not only affect the reduction potential of the cluster but also its electronic structure and spin state.18, 26 For the CrHydA1 C362H variant, the [4Fe-4S]H cluster was found to be stably inserted and the obtained apo-hydrogenase could be artificially maturated with the [2Fe]H cluster precursor. Interestingly, this variant of CrHydA1 displays a higher H2 oxidation activity than the wild type (WT) over a wide range of pH values in protein film electrochemical experiments. However, the H2 oxidation activity requires a moderate overpotential and it cannot reduce protons to hydrogen. In turn we could indeed observe a direct modification of the intermolecular and intramolecular electron transfer pathways induced by the cysteine to histidine exchange in position 362.

The apo-protein was artificially maturated with the [2Fe] complex to generate the active holo-enzyme. Holo-HydA1 C362H had native-like iron content, (SI, Table S1) suggesting full occupancy of the [2Fe]H site in contrast to previous cysteine variants where only partial occupancy of the [2Fe]H site was obtained by in vitro reconstitution.20 The successful integration of the [2Fe]H cluster in the C362H apo-HydA1 variant was further verified by FTIR spectroscopy on the “as isolated” C362H variant, where sharp IR bands arising from the CO and CN- ligands of the integrated [2Fe]H cluster were observed.13 Interestingly, the FTIR spectrum of the “as isolated” C362H variant of CrHydA1 at pH 8 shows mainly Hred and HsredH+, but barely any Hox state (Figure S6), while the WT usually shows a mixture of all three states.20 The position of bands assigned to each of the characteristic redox states are 1 – 3 cm-1 shifted for the C362H variant compared to the WT, as expected for amino acid exchange around the H-cluster.20 When an “as isolated” sample of this variant was oxidized (by adding thionine (E0 = +60 mV) as oxidant) the resulting FTIR spectrum only showed the CO-inhibited state, indicating the destruction of the H-cluster upon oxidation (results not shown). Further attempts to oxidize the sample were also not successful. FTIR measurements at pH 6 were also performed on the “as isolated” CrHydA1 C362H variant (Figure S6). Under these conditions the WT is dominated by

RESULTS AND DISCUSSION The C362H variant of CrHydA1 contains a His-ligated [4Fe-4S] cluster. The cysteine to histidine mutation at the 362 position was performed by site-directed mutagenesis (see experimental section and SI) and the apo-HydA1 C362H variant was recombinantly produced in E. coli. ApoHydA1 C362H had the same iron content as WT, (SI, Table S1) suggesting native-like [4Fe-4S]H cluster assembly. The presence of a preformed [4Fe-4S]H cluster was further confirmed by UV-Vis and EPR spectroscopy. In the UV-Vis spectrum, the intensity and features observed are similar to that of the WT apo-hydrogenase (Figure S1). Apart from the protein backbone peak at 280 nm, a broad shoulder around 420 nm corresponding to the oxidized [4Fe-4S]2+ cluster can be seen. EPR measurements of oxidized (by addition of 1 mM thionine) and reduced samples (by addition of 10 mM sodium dithionite (NaDT)) of the apo-HydA1 C362H variant


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Journal of the American Chemical Society finding could be that the electron transfer from [4Fe-4S]H to [2Fe]H is hindered in this mutant. The imidazole ligand from the His residue is known to withdraw electron density thereby increasing the reduction potential of the [4Fe-4S] cluster and preventing the electron transfer from the [4Fe4S]H to the [2Fe]H cluster. This could have a direct influence on the catalytic behavior of the variant, i.e. on its catalytic bias and overpotential. Therefore, we decided to investigate this further by direct electrochemistry.

the reduced-protonated state of the H-cluster (referred to as HredH+, having its main band at 1891 cm-1).21 This state has a homovalent [Fe(I)Fe(I)] configuration at the binuclear site with a protonated pendant amine, and an oxidized [4Fe-4S]2+ cluster. Conversion of the Hred ([4Fe-4S]H+ - [Fe(I)Fe(II)]) into the HredH+ involves electron transfer from [4Fe-4S]H to [2Fe]H, which is coupled to protonation at the ADT ligand. Surprisingly enough, we did not observe FTIR bands characteristic of HredH+ at pH 6 for the C362H variant, but only the Hred bands (Figure S6). One explanation for this

Figure 1 – Crystal structure of apo-HydA1 C362H (PDB 6GL6; 1.80 Å). A cartoon model of the apo-HydA1 C362H structure and a detailed view of the [4Fe-4S] cluster are shown (magnification). The [4Fe-4S] cluster is coordinated by Cys115, Cys170, Cys366, and the introduced His362 replacing the usual fourth Cys ligation (dist. His-N to Fe 2.18 Å). The Fo - Fc simulated annealing omit map contoured at 3.0 σ is presented as a grey mesh (protein backbone, gray; Fe, brown; S, yellow; C, gray and N, blue). Influence of the His-ligand on the catalytic bias and the overpotential. By protein film electrochemistry, the catalytic activity of the C362H variant was studied at different pH values (Figure 2A) and compared with WT under the same conditions (Figure 2B).The cyclic voltammograms (CVs) for the C362H variant (Figure 2A) are significantly different to the known CVs of [FeFe] hydrogenases as the C362H variant does not produce any H2 at any pH value tested. Furthermore, it displays a large overpotential for H2 oxidation. While the overpotential of WT is slightly pH dependent (it increases slightly with increasing the pH), the pH dependence of the overpotential for the C362H variant is more pronounced.

reversible and the current is recovered by reductive activation with a switch potential and pH dependence similar to the WT (Figure S7). This suggests that this process is coupled to a protonation site that has not been affected by the Cys to His modification. The high potential inactivation has recently been shown to be dependent on the presence of chloride in the solution, which favor the protonation of the ADT.31 The [4Fe-4S] cluster is the gate through which the electrons enter the active site and its redox potential may strongly influence the catalytic bias.32 To oxidize H2, the H-cluster needs to be completely oxidized in the way that it has an open coordination site and it can accept the two electrons from the H2 molecule. Therefore, the catalytic H2 oxidation does not start until the enzyme is fully oxidized in the Hox state. For the WT, this occurs at almost no overpotential, while for the C362H variant the enzyme needs an overpotential of at least 150 mV to be fully oxidized, suggesting a more positive redox potential of the [4Fe-4S] cluster in the C362H variant. Since the thermodynamic potential of H2 at pH 6 is -354 mV, vs SHE,33 the ≈ 150 mV overpotential observed at this pH indicates that the potential of the His-ligated cluster in C362H at pH 6 should be around -200 mV.

The maximum catalytic current for H2 oxidation in C362H is found at pH 6. If we compare the electrocatalytic H2 oxidation activity for the C362H variant and for the WT at this pH, assuming similar coverage for both, the C362H variant of CrHydA1 is three times more active than the WT for H2 oxidation. The higher H2 electrocatalytic current for the C362H variant was reproducible and consistent between different films and batches of the sample. In our previous work, by protein film voltammetry experiments was found that the C362 variants (C362D and C362S) were biased toward H2 oxidation but did not display such a large overpotential, and no variant was more active than the WT.20 The oxidative inactivation at high potential is partly

On the other hand, in order to produce H2, the H-cluster should be completely reduced in the way that it holds the



two necessary electrons to reduce protons to H2 i.e., it should reach the HsredH+ state. Because of the anticipated high potential of the [4Fe-4S] cluster in the C362H variant, electron transfer from the [4Fe-4S] cluster to the [2Fe] cluster is likely to be kinetically limiting during H2 production, whereas this is not the case during H2 oxidation. This explains the incapacity of this enzyme variant to evolve H2.

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and mediator. In contrast the now more positive redox potential of the [4Fe-4S]H cluster in the C362H variant diminishes this reversibility and only allows for oxidation of an electron mediator of a more positive potential compared to MV such as MB. Similarly, solution assays with the natural electron mediator PetF showed no activity, indicating that the variant C362H, is not capable of transferring or accepting electrons from the CrHydA1 native electron mediator PetF, which has a more negative potential (≈ -400 mV vs SHE)34-35 that matches the potential of the WT but not of the C362H variant.

Neither H2 production nor H2 oxidation could be detected in solution assays using methyl viologen (MV) as electron mediator for the CrHydA1 C362H variant. However, when methylene blue (MB) was used as artificial electron acceptor, H2 oxidation could be detected.

Influence of the His-ligand on the reduction potential of the [4Fe-4S] cluster. Besides many other features of the protein structure the nature of the coordinating ligands of the [FeS] cluster is perceived to have the largest influence on its redox potential.17 The presumed shift of the [4Fe-4S]H redox potential, induced by the cysteine to histidine exchange, observed from the electrochemistry was further interrogated. The most direct and efficient way to determine the reduction potential of the cluster would be also through direct electrochemistry by adsorbing the apo-protein on to the electrode surface and measuring the non-turnover signals. However, due to the low coverage of the enzyme on the electrode surface the non-turnover signals in the apohydrogenases could not be detected. In UV-Vis redox titrations using redox mediator dyes the apo C362H variant was found to be unstable over the long course of the experiment, probably due to the mediators. This may also prohibit the use of EPR redox titrations. Therefore, our chosen method to investigate and determine the reduction potential of the His-ligated [4Fe-4S] was spectroelectrochemistry on the holo-enzyme in its CO-inhibited form. It has been already shown for CrHydA1 that spectroelectrochemistry of the CO-inhibited inactive form provides an indirect measurement of the reduction of the [4Fe-4S] cluster in the H-cluster.36 In a spectro-electrochemical experiment, FTIR spectra are recorded over a range of applied potentials. The midpoint potentials of the redox events occurring at the H-cluster can then be obtained from fitting the amplitude of FTIR bands from each of the redox states to the Nernst equation. Figure 3A shows FTIR spectra of the CO-inhibited form of the C362H variant of CrHydA1 at three different potentials measured at pH 8. At the most positive potential (-120 mV, vs SHE) the bands assigned to the Hox-CO state in CrHydA1 can be seen.36 At intermediate potentials (-440 mV, vs SHE) the spectrum resembles that of the Hred-CO state reported by Adamska-Venkatesh et al. for WT CrHydA1, while at negative potential (-720 mV, vs SHE), the small peaks of the HsredH+ state can be detected.36 By following the absorbance of the most intense peak of each state with the applied potential (Figure 3B), it can be seen that the transition between Hox-CO and Hred-CO occurs at -285 mV, about 200 mV more positive than the same transition in WT at the same pH (see Figure 3C, adapted from Adamska-Venkatesh et al.).36

Figure 2 - Comparison of the electrochemical behavior at different pH values for the CrHydA1 C362H variant and CrHydA1 WT. A) Cyclic voltammograms of the C362H variant of CrHydA1 adsorbed onto a rotating pyrolytic graphite electrode and B) cyclic voltammograms of CrHydA1 WT adsorbed onto a rotating pyrolytic graphite electrode, both are measured in buffer mix at different pH values (see methods section), 25 ºC, 2000 rpm rotation rate, 20 mV/s scan rate and under 1 atm H2. The dashed vertical line indicates the reversible hydrogen electrode (RHE) potential to show how the overpotential changes with the pH. The horizontal dashed lines represent the zero current. MB has a more positive redox midpoint potential compared to MV (Em= +11 mV, vs SHE for MB compared with -446 mV, vs SHE for the MV). In the WT the redox potential of the H-cluster is close to the midpoint potential of MV allowing for a reversible electron transfer between enzyme

CO bound to the open coordination site in the CO-inhibited enzyme should slightly increase the electron density in the H-cluster, hence lowering the redox potential of the [4Fe-4S]


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Journal of the American Chemical Society (Figure 1). Under physiological conditions (pH ≈ 7.4), the non-ligated N-His (which is more exposed to the protein surface) will be singly protonated and the imidazole ring will be uncharged (in contrast to the negatively charged thiolate ligands), hence facilitating electron withdrawal from the cluster and increasing its reduction potential. Deprotonation of free histidine in solution to the imidazolate anion form occurs at pH values greater than 14. However, the imidazolate anion is stabilized by ligation to Fe shifting the pKa value into the physiological range.38-39 The protonated N-His could also form H-bonding interactions with the solvent and/or protein residues further modifying the pKa value and hence the pH dependence of the redox potential of the [4Fe-4S]H.11

cluster.36-37 However, the difference between the potential of the WT and the C362H variant in the CO-inhibited and active states should be the same. As has been already demonstrated, the transition between Hox-CO and Hred-CO involves reduction of [4Fe-4S]H. At more negative potentials, after the Hred-CO is formed, a second reduction occurs. This second electron has to be placed at the [2Fe] sub-cluster. Reduction of the [2Fe] is coupled to the protonation of the bridging ADT ligand and the excess of electron density facilitates the CO release, hence generating the HsredH+ state. Histidine coordination of [4Fe-4S]H is achieved through a bond between one N-His and an iron ion from the cluster

Figure 3 – Comparison of the [4Fe-4S]H reduction potential of the C362H variant of CrHydA1 and CrHydA1 WT measured by CO-inhibited spectro-electrochemistry. A) Selected FTIR spectra recorded at three different potentials from the spectroelectrochemical redox titration of 1 - 2 mM of CO-inhibited CrHydA1 C362H variant in 100 mM Tris-HCl + 150 mM KCl at pH 8 and at 15 ºC. The selected spectra at -120 mV, -440 mV and -720 mV are shown to highlight the Hox-CO (light blue), Hred-CO (dark blue) and HsredH+ (green) states. B) Plot representing the change in FTIR absorbance with redox potential for the C362H CrHydA1 variant. The mean bands of the Hox-CO (2015 cm-1), Hred-CO (2005 cm-1) and HsredH+ (1884cm-1) states are plotted against the applied potential. The solid lines represent the n = 1 Nernstian fittings. C) Reductive titration of the CO-inhibited WT CrHydA1at pH 8 and 15 ºC adapted from Adamska-Venkatesh et al.36 The change in the FTIR absorbance of the mean peak from each state Hox-CO (2013 cm-1), Hred-CO (1793 cm-1) and HsredH+ (1882 cm-1) is plotted against the redox potential. The solid lines represent the n = 1 Nernstian fittings. All potentials are quoted versus the standard hydrogen electrode (SHE). Spectro-electrochemical redox titrations were performed in the reductive direction. The subsequent oxidative titrations showed similar behavior but lower signal intensity due to some degradation of the sample over the long time-course of the experiments.



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Influence of the His-ligand on the overall catalytic mechanism: formation of the hydride state. To date, five redox states of the H-cluster have been spectroscopically identified for CrHydA1, giving an almost complete picture of the potential catalytic cycle.21-22, 41-44 The oxidized state (Hox) with an oxidized [4Fe-4S]H cluster and a mixed valence [Fe(I)Fe(II)] in the [2Fe]H sub-cluster, gets one electron reduced (the reduction occurs at [4Fe-4S]H) generating the Hred state ([4Fe-4S]H+ - [Fe(I)Fe(II)]). Protonation of the ADT moiety is coupled to electron transfer from [4Fe-4S]H to [2Fe]H yielding the HredH+ state ([4Fe-4S]H2+ [Fe(I)Fe(I)]).21 This enables a second reduction at [4Fe-4S]H providing the HsredH+ state,41, 44 which may rearrange to form a hydride-bound state, Hhyd, with an [Fe(II)Fe(II)] electronic configuration at the binuclear site and a reduced [4Fe-4S]H.22, 45-46 A second protonation could potentially be coupled to electron transfer to [2Fe]H, forming H2 and regenerating the initial Hox state (Scheme 1). The Hhyd state is proposed to be a key intermediate in [FeFe]-hydrogenase catalysis. The protonation of an iron-bound terminal hydride via proton transfer from the bridgehead nitrogen of the protonated ADT to the Fed ion holding the hydride, may be the final step in H2 formation. The role of PCET in the catalytic mechanism of [FeFe] hydrogenases is essential to avoid the accumulation of hydride intermediates.22

In order to see if a pH dependence can be observed, spectroelectrochemical redox titrations at different pH values were performed for the C362H variant and for WT. Figure S8 shows the spectro-electrochemical redox titration for both the C362H variant (Figure S8. A and B) and the WT (Figure S8. C and D) at pH 6 (Figure S8. A and C) and at pH 10 (Figure S8. B and D). The Hox-CO / Hred-CO transition, associated with the reduction of [4Fe-4S]H is 95 mV more positive at pH 6 than at pH 8. However, this transition at pH 10 has the same potential (within the experimental error, ± 25 mV) than at pH 8 (Table S2). The difference between the H2 thermodynamic potential and the reduction potentials of [4Fe-4S]H determined by spectroelectrochemistry at various pH values agrees with the overpotentials observed in electrochemistry (Figure 2). This shows how the findings from the electrochemistry are nicely correlated with the results obtained by FTIR spectroelectrochemistry. It was essential to check whether similar pH dependence could be observed for WT CrHydA1. However, for WT, the pH dependence was more complicated. At pH 6, protonation of the Hred-CO state is favorable leading to coupled electron transfer from [4Fe-4S]H to [2Fe]H followed by release of CO to form HredH+ (around -300 mV). At more negative potential (-450 mV), HsredH+ appears at the same time as Hred-CO (see supplementary discussion and Figure S9).

Hhyd can be produced in [FeFe]-hydrogenase at low pH (≤ 6) under strong reducing conditions (100 mM NaDT) and in the presence of H2.47 As shown in Figure 4, the formation of the Hhyd state under these conditions was successful for the C362H variant. The bands describing the hydride state in this mutant are 2088 cm-1 and 2077 cm-1 for the CN- ligands, 1981 cm-1 and 1961 cm-1 for the terminal CO ligands and 1865 cm-1 for the bridging CO.

At pH 10 the behavior of the CO-inhibited WT is very similar to that at pH 8 previously reported by AdamskaVenkatesh et al.36 having the same potential (within the experimental error, ± 25 mV) for the Hox-CO / Hred-CO transition. At pH 10, both C362H variant and WT, were not stable, especially at negative potentials, so the complete formation of the HsredH+ cannot be observed. The potential for the Hred-CO / HsredH+ transition in the C362H variant shows a pH dependence between pH 6 and 8 (Table S2). This transition should be coupled to protonation at the ADT site for both the WT and the C362H variant. The value determined for the WT at pH 6 may be distorted by the change of pH during the measurement (see supplementary discussion). This distortion was prevented by the high potential of the [4Fe-4S] cluster of the C362H variant. In the sensory hydrogenase from Thermotoga maritima, the [2Fe] site has a higher potential causing electron transfer to this site in the absence of protonation. In the C362H variant of CrHydA1, the opposite is observed as the [4Fe-4S]H cluster potential is more positive, preventing electron transfer to [2Fe] even when protonation should be favorable.40 The positive potential of the [4Fe-4S] cluster disrupts the electron transfer between the two sub-clusters of the Hcluster and forces [4Fe-4S]H to be always reduced, explaining the predominance of the Hred state observed in the “as isolated” samples. As observed in electrochemistry, the disruption of the electron transfer between the two subclusters of the H-cluster as a consequence of the high potential of [4Fe-4S]H has a big influence on the catalytic behavior of the CrHydA1 C362H variant. In order to see how the catalytic behavior of C362H correlates with the catalytic cycle our next step was to investigate the formation of the hydride state in this variant.

Figure 4 – Formation of the hydride state in the CrHydA1 C362H variant at pH 6. FTIR spectra of CrHydA1 C362H variant (200 µM in 100 mM MES, 300 mM NaCl, pH 6) with addition of 100 mM sodium dithionite under atmosphere of H2 to form the hydride state. The main peaks of the hydride state are colored in red. The nonassigned bands in the spectra correspond to small contributions from the HsredH+, Hred-CO and Hox states.

6

All the bands are slightly blue shifted (1 – 4 cm-1) relative to those in the Hhyd state from WT (Figure S10); interestingly

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the bridging CO is the most affected (4 cm-1). The stability of the Hhyd state was followed by collecting spectra over time. Only about 40% of the intensity of the Hhyd bands was lost after 50 hours compared with 60% for WT (Figure S10). Interestingly, WT under these conditions contains a small amount of a state called Hox-blue (it has a spectral signature similar to Hox but shifted to higher energy, with a main band at 1947 cm-1).

the expense of displaying a large overpotential. This highlights the importance of tuning the potential of electrontransfer centers for catalysis. In the [FeFe] hydrogenases, the potential of the [4Fe-4S] cluster directly influences the proton-coupled electron transfer (PCET) processes in the Hcluster, which alters the catalytic properties of the enzyme (Scheme 1). The reduction potential of the cluster as well as the overpotential for H2 oxidation is altered due to the incorporated His-ligand. In a broad context, the work presented here gives insights into how [FeS] clusters work as a redox centers in catalysis. It is important to gain fundamental knowledge in understanding and interpreting the function of [4Fe-4S] clusters. Bioenergetics processes such as photosynthesis and respiration require electron transfer between different redox partners. Iron-sulfur (FeS) proteins are one of the most widely used and more efficient electron-transfer centers in biology.48-49 Understanding how they function as gate/bridge for mediating electron transfer reactions between electron carriers and catalytic sites is of relevant interest to designing efficient synthetic catalysts and/or artificial metalloenzymes for energy conversion.

The Hox-blue state is thought to be protonated at Cys362.23, 25 C362H appears to contain no traces of Hox-blue under these conditions. These data suggest that indeed the Hhyd state can be formed in the C362H variant. However, a second protonation coupled to electron transfer from the reduced [4Fe-4S] cluster to [2Fe]H is needed in order to form H2 and this electron transfer is probably hindered in this variant due to the potential difference between the two sites. This may be part of the reason why this variant does not produce H2: it may get stuck in the Hhyd state due to kinetically limited electron transfer from the [4Fe-4S] cluster to the [2Fe] cluster.

EXPERIMENTAL SECTION Generation of the C362H HydA1 variant. Site-directed mutagenesis at position C362 (numbering according to the expressed amino acid sequence) was performed by QuikChange using pET21(b)-hydA150 as the template plasmid and the mutagenic primers listed on Table S2. Introduction of the desired mutation was verified by sequencing. Protein overexpression and in vitro maturation. [FeFe] hydrogenase HydA1 from C. reinhardtii (48 kDa) with Cterminally fused Strep-tagII and its C362H variant were heterologously expressed in the E. coli strain BL21(DE3)ΔiscR51 in the absence of specific maturases, hence lacking the [2Fe] sub-cluster (apoHydA1).50 Streptactin affinity chromatography (IBA, Göttingen, Germany) was used for protein purification under strictly anaerobic conditions using 100 mM Tris–HCl buffer (pH 8) containing 2 mM NaDT.52 Protein preparations used for UV-Vis spectroscopy and spectro-electrochemistry were purified without NaDT. Protein yields were about 5 to 12 mg for the C362H variant out of 1 L expression culture compared to up to 30 mg for the WT. Protein concentrations were determined by the method of Bradford53 using bovine serum albumin as a standard and protein purity was assessed by SDS-PAGE (see Figure S10). Bovine serum albumin was obtained commercially from Carl Roth, Karlsruhe, Germany. [Fe2[µ-(SCH2)2NH](CN)2(CO)4][Et4N]2 ([2Fe]ADT) was prepared as described previously27 and dissolved in 100 mM K2HPO4/KH2PO4 buffer (pH 6.8) to concentrations of 21 to 55 mM. A 5-fold molar excess of [2Fe]ADT was added to 200 to 400 μM apoHydA1 in a final volume of up to 500 µL 100 mM K2HPO4/KH2PO4 buffer (pH 6.8) with 2 mM NaDT initiating the maturation process at 25 °C for 1 h.14 Subsequently, the excess [2Fe]ADT was removed via size exclusion chromatography using a NAP-5 column (GE Healthcare, Chicago, USA) equilibrated with 100 mM Tris– HCl (pH 8) and 2mM NaDT. Protein preparations were concentrated up to 2 mM using 30 kDa Amicon Ultra

Scheme 1 – Proposed catalytic cycle and CO inhibition/release for the CrHydA1 C362H variant. The large green arrows indicate the steps that are expected to be favored in the C362H variant compared with WT while the small blue arrows indicate the steps expected to be hindered for C362H compared with WT. The Hox-CO and Hred-CO states of the CO inhibited enzyme are also shown. CONCLUSIONS In this work, we have demonstrated that the redox potential and properties of the H-cluster can be elegantly tuned by site-differentiated amino acid exchange around the coordination sphere of the [4Fe-4S] cluster. We have studied in detail the effects of exchanging the coordinating cysteine 362 to a histidine ligand on the redox and structural properties of the [4Fe-4S] cluster. The properties of the resulting C362H variant of CrHydA1 are dramatically modified, being absolutely biased towards H2 oxidation at

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transferred to the surface of the electrode and allowed to adsorb for 5 min. Prior to the measurement, the nonadsorbed protein was removed from the electrode surface by rinsing it with the corresponding buffer. The electrochemical measurements were performed in a standard three-electrode electrochemical cell with a saturated calomel reference electrode (SCE) separated from the main compartment in a sidearm containing 0.1 M KCl. The conditions used for the electrochemical experiments were: 25 ºC, buffer mix at each different pH value, 20 mV s-1, 1 atm H2 (1 L min-1 total flow) and 2000 rpm electrode rotation rate to avoid mass-diffusion limitation. The potential was controlled by a PARSTAT MC-1000 multi-channel potentiostat (Princeton Applied Research, Oak Ridge, USA). The reference electrode potential was periodically controlled using (hydroxymethyl) ferrocene (+420 mV vs. SHE).58

centrifugal filter units (Merck Millipore, Billerica, USA) and stored anaerobically at - 80 °C. Iron quantification. Iron content of samples was quantified in triplicate using the method of Fish54 and protein concentrations were determined in parallel. Table S1 summarizes the results. UV-Vis spectroscopy. UV-Vis spectra were recorded with a UV-2450 spectrometer (Shimadzu, Kyoto, Japan) at 25 °C using sealed 1 mL UV cuvettes filled with 500 mL of apoHydA1 solution at a concentration of 10 μM in 100 mM Tris–HCl buffer (pH 8). EPR. X-band (≈ 9.6 GHz) EPR samples (200 µL) were prepared in an anaerobic glovebox filled with N2 and transferred to 4 mm (o.d.) quartz EPR tubes, frozen in liquid N2. For reduced samples, 10 mM sodium dithionite was added, while for oxidized samples 1 mM thionine was added. The measurements were performed on a Bruker ELEXSYS E500 CW X-band EPR spectrometer with an Oxford Instruments ESR900 helium flow cryostat connected to an ITC503 temperature controller. The parameters used were: microwave frequency 9.64 GHz, time constant 81.92 ms, conversion time 81.92 ms, modulation frequency 100 kHz. All other parameters varied and are given in the figure legends. EPR spectral simulations were performed using Easyspin.

Solution activity assays. For determination of the methyl viologen (MV) dependent H2 evolution and uptake, standard in vitro enzymatic activity assays were performed as described elsewhere.20 The solution assays with the natural redox partner, PetF, were performed as described previously.20 The hydrogen uptake determination using methylene blue (MB) as electron acceptor was based on methods already described in the literature.59-60 1 μg of protein was transferred into a sealed UV/Vis cuvette and purged with 100% H2. Next, 1 mL of 50 μM methylene blue solution in 100 mM Tris-HCl (pH 8) saturated with H2 was added to the cuvette. Discoloration of the methylene blue solution was recorded with a UV-2450 spectrometer (Shimadzu, Kyoto, Japan) at 25 °C and activity was estimated based on the initial slope of absorbance vs time using linear regression. The H2 oxidation by the enzyme can be monitored photometrically at λ= 660 nm following the reduction of the dark blue methylene blue solution which discolors over time (oxidized state: blue, reduced state: colorless).

Crystallography. A 1:1 mixture of 200 μM apoHydA1 C362H and 100 mM MES pH 6.0, 0.6 M NaCl, 12%, PEG 4000 buffer was used in a hanging drop vapor diffusion experiment leading to brown crystals within 6 days at 4 °C (see Figure S3). Crystals were mounted into Cryo-Loops™ (Hampton Research) and flash frozen with liquid N2 under anaerobic conditions. Collection of diffraction data took place at the ESRF ID30B beamline (Grenoble, France) at 100 K with incident beam energy of 12,700 eV. The data-set was processed with the space group P 32 2 1 using the software package XDS55 and structure optimization was performed with the help of software packages PHENIX56 and COOT.57 The available structure of another apoHydA1 variant (PDB 6GM5) with a resolution of 1.45 Å was used as starting model for refinement (see SI, Table S3 for crystal data and refinement statistics). This led to a model refined to 1.80 Å resolution with Rwork and Rfree values of 0.1694 and 0.1968 (PDB 6GL6), respectively. Simulated annealing omit maps were calculated with PHENIX, omitting the [4Fe-4S] cluster and its coordinating residues (C115, C170, H362 and C366) as well as the slightly shifted residues (C170, T171 and R172) and the SO32- ion in close proximity to the [4Fe4S] cluster (Fig. S5).

Spectro-electrochemistry. Spectro-electrochemical redox titrations were performed using a home-built electrochemical FTIR-cell, designed according to its original design by Moss and co-workers.44, 61 The experiments were carried out in a Bruker IFS 66v/S FTIR spectrometer equipped with a nitrogen cooled Bruker mercury cadmium telluride (MCT) detector. Under anaerobic conditions inside a Glove-box filled with N2 (MBRAUN, Garching, Germany), samples (35 - 40 µL) of 1 - 2 mM C362H and WT of CrHydA1 in buffers 100 mM MES + 300 mM KCl pH 6, 100 mM TRIS-HCl + 300 mM KCl pH 8 or 100 mM CAPS + 300 mM KCl pH 10 were placed between CaF2 windows on a gold mesh (≈ 50 μm thick), which functions as a working electrode. A platinum counter electrode and an Ag/AgCl (sat. KCl) reference electrode were also placed in the built electrochemical FTIR-cell, forming the three-electrode system. The reference electrode was calibrated before and after each measurement using (hydroxymethyl)ferrocene (Aldrich, +420 mV vs SHE) as a reference, to ensure the potential stability during the course of the experiment. The potential was regulated by an Autolab PGSTAT101 potentiostat using Nova software with an equilibration time of 90 min at each potential. Spectra were collected in the double-sided, forward-backward mode with a resolution of 2 cm-1 or 0.2 cm-1, an aperture setting of 1.5 mm and a scan velocity of 20 Hz. The temperature of the cell was

Protein film electrochemistry. A pyrolytic graphite rotating electrode (0.031 cm2 homemade pyrolytic graphite from Momentive Materials) was polished with alumina MasterPrep Polishing Suspension (0.05 mm, Buehler, Esslingen, Germany) and sonicated for 5 min in Milli-Q water. Afterwards, the electrode was transferred into the N2 Glove-box (MBRAUN, Garching, Germany) to remove surface-adsorbed oxygen by 10 potential cycles from +250 mV to -650 mV, vs SHE in a buffer a mix at pH 7 containing 15 mM MES, HEPES, TAPS, CHES and sodium acetate and 0.1 M NaCl. For the protein adsorption, 4 μL of 6 µM protein in 10 mM MES pH 5.8 + 2 mM NaDT were

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maintained using a water circulator system (Huber, Offenburg). Data analysis was performed using homewritten routines in the MATLABTM environment. All other parameters are detailed in the figure legends. All potentials referred to in the text are quoted versus the standard hydrogen electrode (SHE) unless it is otherwise specified.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Supplementary discussion, UV-Vis, EPR, photos of apo-HydA1 C362H crystals, structural overlay of apo-HydA1 C362H and apo-HydA1 WT, simulated annealing omit map of apo-HydA1 C362H, spectro-electrochemical redox titrations for C362H and WT at pH 6 and pH 10, comparison of the pH dependence of the switch potential for C362H and WT, FTIR time course for Hhyd in the C362H variant, SDS-PAGE gel apo-HydA1 C362H variant, included in Figures S1−S11 and Table S1-S3.

AUTHOR INFORMATION

[4Fe-4S]H

[4Fe–4S] cluster of the H-cluster

Apo-HydA1

Apoform of HydA1 only harboring the [4Fe–4S] subcluster

C. reinhardtii

Chlamydomonas reinhardtii

EPR

Electron paramagnetic resonance

Fed

Distal iron atom of the [2Fe] cluster relative to the [4Fe–4S] moiety

FTIR

Fourier-transform infrared

Holo-HydA1

Holoform of HydA1 harboring [4Fe–4S]+ [2Fe] sub-cluster

MV

Methyl viologen

MB

Methylene blue

NaDT

Sodium dithionite

SHE

Standard hydrogen electrode

RHE

Reversible hydrogen electrode

WT

Wild-type enzyme

the

Corresponding Authors [email protected] [email protected]

REFERENCES 1. Jones, A. K.; Sillery, E.; Albracht, S. P. J.; Armstrong, F. A., Direct Comparison of the Electrocatalytic Oxidation of Hydrogen by an Enzyme and a Platinum Catalyst. Chem. Commun. 2002, 866-867. 2. Vignais, P. M.; Billoud, B., Occurrence, Classification, and Biological Function of Hydrogenases: An Overview. Chem. Rev. 2007, 107, 4206-4272. 3. B. R. Glick, W. G. M., and S. M. Martin, Purification and Properties of the Periplasmic Hydrogenase from Desulfovibrio Desulfuricans. Can. J. Microbiol. 1980, 26, 1214-1223. 4. Armstrong, F. A.; Hirst, J., Reversibility and Efficiency in Electrocatalytic Energy Conversion and Lessons from Enzymes. Proc. Natl. Acad. Sci. USA 2011, 108, 1404914054. 5. Adams, M. W. W., The Structure and Mechanism of IronHydrogenases. Biochim. Biophys. Acta 1990, 1020, 115-145. 6. Orain, C.; Saujet, L.; Gauquelin, C.; Soucaille, P.; Meynial-Salles, I.; Baffert, C.; Fourmond, V.; Bottin, H.; Léger, C., Electrochemical Measurements of the Kinetics of Inhibition of Two FeFe Hydrogenases by O2 Demonstrate That the Reaction Is Partly Reversible. J. Am. Chem. Soc. 2015, 137, 12580-12587. 7. Fourmond, V.; Greco, C.; Sybirna, K.; Baffert, C.; Wang, P.-H.; Ezanno, P.; Montefiori, M.; Bruschi, M.; MeynialSalles, I.; Soucaille, P.; Blumberger, J.; Bottin, H.; De Gioia, L.; Leger, C., The Oxidative Inactivation of FeFe Hydrogenase Reveals the Flexibility of the H-Cluster. Nat. Chem. 2014, 6, 336-342. 8. Kubas, A.; Orain, C.; De Sancho, D.; Saujet, L.; Sensi, M.; Gauquelin, C.; Meynial-Salles, I.; Soucaille, P.; Bottin, H.; Baffert, C.; Fourmond, V.; Best, R. B.; Blumberger, J.; Léger, C., Mechanism of O2 Diffusion and Reduction in FeFe Hydrogenases. Nat. Chem. 2017, 9, 88-95.

Author Contributions #PRM and LK contributed equally to the experimental work.

Notes The authors declare no competing financial interest.

Funding Sources This work was financially supported by the Max Planck Society and by the Cluster of Excellence RESOLV (EXC1069) from the Deutsche Forschungsgemeinschaft (DFG). The authors are grateful to Volkswagen Foundation (LigH2t) and the DIP Programme (LU 315/17-1) funded by the Deutsche Forschungsgemeinschaft (DFG). LK was supported by a Kekulé Mobility Fellowship from the the Fond der Chemischen Industrie.

ACKNOWLEDGMENT We kindly thank Florian Wittkamp and Ulf-Peter Apfel for providing the [2Fe]ADT used for the in vitro maturation of apoHydA1. The authors would like to also thank Nina Breuer for help with preparation of CrHydA1 samples as well as Birgit Nöring for her technical support in protein-film electrochemical experiments.

ABBREVIATIONS ADT

aza-propane-1,3-dithiolate

[2Fe]H

[2Fe–2S] sub-cluster of the H-cluster

[2Fe]ADT

Chemically analogue

synthesized

[2Fe]

cluster

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23. Senger, M.; Laun, K.; Wittkamp, F.; Duan, J.; Haumann, M.; Happe, T.; Winkler, M.; Apfel, U.-P.; Stripp, S. T., Proton-Coupled Reduction of the Catalytic [4Fe-4S] Cluster in [FeFe]-Hydrogenases. Angew. Chem. Int. Ed. 2017, 56, 16503-16506. 24. Haumann, M.; Stripp, S. T., The Molecular Proceedings of Biological Hydrogen Turnover. Acc. Chem. Res. 2018, 51, 1755-1763. 25. Senger, M.; Mebs, S.; Duan, J.; Shulenina, O.; Laun, K.; Kertess, L.; Wittkamp, F.; Apfel, U.-P.; Happe, T.; Winkler, M.; Haumann, M.; Stripp, S. T., Protonation/ Reduction Dynamics at the [4Fe-4S] Subsite of the Hydrogen-Forming Cofactor in [FeFe]-Hydrogenases. Phys. Chem. Chem. Phys., 2018, 20, 3128-3140. 26. Adamson, H.; Robinson, M.; Wright, J. J.; Flanagan, L. A.; Walton, J.; Elton, D.; Gavaghan, D. J.; Bond, A. M.; Roessler, M. M.; Parkin, A., Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase Via a Single Amino Acid Exchange at the Electron Entry/Exit Site. J. Am. Chem. Soc. 2017, 139, 10677-10686. 27. Esselborn, J.; Muraki, N.; Klein, K.; Engelbrecht, V.; Metzler-Nolte, N.; Apfel, U. P.; Hofmann, E.; Kurisu, G.; Happe, T., A Structural View of Synthetic Cofactor Integration into [FeFe]-Hydrogenases. Chem. Sci. 2016, 7, 959-968. 28. Kertess, L.; Wittkamp, F.; Sommer, C.; Esselborn, J.; Rüdiger, O.; Reijerse, E. J.; Hofmann, E.; Lubitz, W.; Winkler, M.; Happe, T.; Apfel, U. P., Chalcogenide Substitution in the [2Fe] Cluster of [FeFe]-Hydrogenases Conserves High Enzymatic Activity. Dalton Trans. 2017, 46, 16947-16958. 29. Lin, J.; Zhou, T.; Ye, K.; Wang, J., Crystal Structure of Human Mitoneet Reveals Distinct Groups of Iron–Sulfur Proteins. Proc. Natl. Acad. Sci. USA 2007, 104, 14640-5. 30. Mulder, D. W.; Boyd, E. S.; Sarma, R.; Lange, R. K.; Endrizzi, J. A.; Broderick, J. B.; Peters, J. W., Stepwise [FeFe]-Hydrogenase H-Cluster Assembly Revealed in the Structure of HydaΔefg. Nature 2010, 465, 248-252. 31. del Barrio, M.; Sensi, M.; Fradale, L.; Bruschi, M.; Greco, C.; de Gioia, L.; Bertini, L.; Fourmond, V.; Léger, C., Interaction of the H-Cluster of Fefe Hydrogenase with Halides. J. Am. Chem. Soc. 2018, 140, 5485-5492. 32. Hexter, S. V.; Grey, F.; Happe, T.; Climent, V.; Armstrong, F. A., Electrocatalytic Mechanism of Reversible Hydrogen Cycling by Enzymes and Distinctions between the Major Classes of Hydrogenases. Proc. Nat. Acad. Sci. USA 2012, 109, 11516-11521. 33. Vincent, K. A.; Parkin, A.; Armstrong, F. A., Investigating and Exploiting the Electrocatalytic Properties of Hydrogenases. Chem. Rev. 2007, 107, 4366-4413. 34. Liu, J., Metalloproteins Containing Cytochrome, Iron– Sulfur. Chem. Rev. 2014, 114, 4366-469. 35. Terauchi, A. M.; Lu, S. F.; Zaffagnini, M.; Tappa, S.; Hirasawa, M.; Tripathy, J. N.; Knaff, D. B.; Farmer, P. J.; Lemaire, S. D.; Hase, T.; Merchant, S. S., Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii. J. Biol. Chem. 2009, 284, 25867-78. 36. Adamska-Venkatesh, A.; Krawietz, D.; Siebel, J.; Weber, K.; Happe, T.; Reijerse, E.; Lubitz, W., New Redox States Observed in [FeFe] Hydrogenases Reveal Redox Coupling within the H-Cluster. J. Am. Chem. Soc. 2014, 136, 11339-11346.

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[FeFe] hydrogenase - American Chemical Society

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