Interplay between CNâ Ligands and the Secondary Coordination

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Interplay between the CN-ligands and the secondary coordination sphere of the H-cluster in [FeFe]-hydrogenases Oliver Lampret, Agnieszka Adamska-Venkatesh, Hannes Konegger, Florian Wittkamp, Ulf-Peter Apfel, Edward J. Reijerse, Wolfgang Lubitz, Olaf Rüdiger, Thomas Happe, and Martin Winkler J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08735 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Journal of the American Chemical Society

Interplay between the CN--ligands and the secondary coordination sphere of the H-cluster in [FeFe]-hydrogenases Oliver Lampret1 & Agnieszka Adamska-Venkatesh2, Hannes Konegger,1 Florian Wittkamp,3 UlfPeter Apfel,3 Edward J. Reijerse,2 Wolfgang Lubitz,2 Olaf Rüdiger,*,2 Thomas Happe*,1 Martin Winkler*,1 1

Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany 2

Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim an der Ruhr, Germany

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Fakultät für Chemie und Biochemie, Lehrstuhl für Anorganische Chemie I-Bioanorganische Chemie, Ruhr Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany

Abstract: The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4FeH) linked to a binuclear iron sulfur cluster (2FeH) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2FeH) subsite features a diatomic coordination sphere composed of three CO and two CN- ligands affecting its redox properties and providing excellent probes for FTIR-spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN- signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN- ligands tightly anchoring the (2FeH) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN- ligands on FTIRspectroscopy and catalysis. By comparing the FTIR-spectra of wild type enzyme and two mutagenesis variants we are able to assign the IR signals of the individual CN- ligands of the (2FeH) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN- ligand (i.e. closest to the (4FeH) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

1. INTRODUCTION -1

With turnover rates of up to 10000 s for the reversible conversion of protons and electrons to molecular hydrogen (H2), [FeFe]-hydrogenases are promising targets for the development of sustainable hydrogen catalysts.1-3 Since the industrial applicability for [FeFe]hydrogenases is still out of reach, researchers during the last decade have strived for the development of efficient hydrogenase biomimics.4-7 The basis for this approach is a fundamental understanding of the H-cluster, the protein’s catalytic center and its synergy with the protein environment. HydA1, the ferredoxin-dependent [FeFe]hydrogenase from Chlamydomonas reinhardtii (CrHydA1) is the best characterized example of the least complex structured M1 subtype which harbors no additional iron-sulfur clusters besides the H-cluster.8-10 The H-cluster is embedded in a mainly hydrophobic pocket

within the protein scaffold of the H-domain which is more or less conserved among all [FeFe]-hydrogenases.1112 Understanding the role of the polypeptide environment around the H-cluster remains a major challenge in [FeFe]-hydrogenase research. The H-cluster is composed of a “standard” cubane [4Fe-4S]-cluster (4FeH) which is linked through a bridging cysteine thiol group to the binuclear iron sulfur moiety ((2FeH) cluster), whose two Fe-centers are coordinated by two nonproteinogenic CN- and three CO ligands (see Figure 1).1314 They stabilize the low spin and low oxidation states of the Fe atoms needed for the H+/H2 turnover process.15-18 The peptide environment of the (2FeH) cluster is strongly conserved among [FeFe]-hydrogenases indicating its importance in the preservation of a functional ligand configuration and a stable cofactor integration into the binding pocket.9, 19

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Figure 1: Stick-model of the H-cluster embedded into the protein environment. A) H-cluster of CpI (PDB ID: 4XDC) and Hbond network with its protein environment. B) Modeled structure of the H-cluster from CrHydA1 (PDB ID: 3LX4) with in silico integration of the (2FeH) subcluster based on the H-cluster coordinates of CpI (4XDC). The corresponding H-bonds of the two networks that coordinate the (2FeH) ligand within the protein environment are indicated by dashed lines. A comparison of the secondary ligand spheres in both structures reveals a high degree of homology in sequence and structure. One additional Hbond can be observed for the CNP ligand in CpI originating from Ser232 (Ala94 in CrHydA1) as H-bond donor. Relevant amino acid positions for site directed and saturation mutagenesis experiments (S232Cp, A92Cr, A94Cr, S193Cr and E231Cr) are highlighted by a black carbon backbone. Grey/Black: Carbon; Blue: Nitrogen; red: Oxygen, Yellow: Sulfur; Orange: Iron

Apart from providing substrate (H+/H2) transfer channels, the peptide-based secondary sphere also stabilizes both CN- groups (CNp and CNd, the subscript p or d refers to its position to the (4FeH), being proximal, p or distal, d) in trans configuration by two precisely arranged hydrogen bond networks.9, 20 H-bonding to the CN- ligands is further believed to facilitate formation of the open coordination site at Fed which is essential for fast catalytic turnover.21-22 Incorporation of the binuclear (2FeH) site containing only one CN- ligand was shown to be possible, however at the expense of stability and activity, thus emphasizing the high importance of Hbond contacts to both CN- ligands.23 For the [FeFe]hydrogenase I from Clostridium pasteurianum (CpI), several amino acids have been suggested to be involved in H-bonding to the (2FeH) cluster (see Figure 1).24 In CrHydA1 homologous amino acids are assumed to fulfill the same H-bonding functions with the exception of Ser232, which in CrHydA1 corresponds to Ala94. Given its close proximity (3.1 Å) to CNP in CpI, the hydroxyl group of S232 could influence both the coordination and electronic structure of the (2FeH) cluster. Figure 1 shows the H-cluster in the crystal structure of CpI and a structure model of CrHydA1, including the amino acid positions that form the individual environments of both CNligands. It is important to note that the H-bond interactions can be formed by the NH-groups from the amino acid backbone as well as from their side chain groups. The cofactor stabilizing influence of peptide based Hbond partners of CNd became apparent upon mutagenesis at position K358Cp (K228Cr) which yielded enzyme variants (e.g. K358CpN) incapable of incorporating the (2FeH) subcluster.19 However, the influence of other

individual H-bonds on the catalytic behavior of the Hcluster, on its redox- and spectroscopic features remains to be investigated. Fourier transform infrared (FTIR) spectroscopy provides a direct window into the active site of [FeFe]hydrogenases. The pattern of CO and CN- stretches originating from the (2FeH) site can be interpreted in terms of molecular structure and redox properties of the H-cluster.25-27 More importantly, it is an invaluable technique for monitoring the fractions of individual catalytic and non-catalytic states which feature characteristic CO and CN- vibrational signatures.28-29 Changes in the redox states of the (2FeH) subsite are reflected by characteristic vibrational modes of the CN− and CO ligands.27 For the catalytic cycle several H-cluster states have been proposed to be relevant. Most importantly, the oxidized active ready state Hox (mixed valence binuclear Fe(I)Fe(II) core and oxidized [4Fe-4S]2+ cluster) undergoes a fully reversible consecutive two-step electron reduction process to the two reduced states Hred (reduction on the (2FeH) subsite (Fe(I)Fe(I)), or [4Fe4S]+ cluster) and the “superreduced” state Hsred (additional reduction to [4Fe-4S]+ Fe(I)Fe(I) form), respectively.30-31 Recently, we and others were able to accumulate and spectroscopically characterize the presupposed Hhyd state which carries a terminal hydride species for active wild type enzyme and enzyme variants affected in proton transfer.29, 32-33 Additionally, extrinsic CO can reversibly bind to Hox and Hred forming the inhibited states Hox-CO and Hred-CO.27 The assignment of individual vibrational frequencies originating from the CO ligands has already been accomplished by a combination of 13CO exposure and

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illumination steps at different wavelengths, exploiting the photo-lability of the CO ligands.34 However, the assignment of individual CN- ligand bands was assumed to be impossible due to the high symmetry of the binuclear site which prevents site selective 13CN- or C15N labeling within the H-cluster.35 In the present work, we direct our attention to the specific H-bonding partners of the individual CN- ligands from the protein surrounding. We show that targeted mutations enable the assignment of the two state specific CN- FTIR bands to the individual ligands.

2. EXPERIMENTAL SECTION Plasmid Preparation. CrHydA1 mutants were prepared, using the QuikChange method following an inhouse adapted protocol.36 The parental plasmid pET21b bearing the Escherichia coli codon optimized wild type (WT) gene hydA1 was amplified by PCR using Phusion DNA polymerase (ThermoFisher) together with 2025 bp primers. The PCR product was digested for 2 h at 37°C with restriction enzyme DpnI (NEB). Chemically competent E. coli cells of strain DH5α were transformed afterwards. All positive mutants were confirmed by sequencing (RUB sequence service). Enzyme Expression and Purification. Recombinant apoproteins of CrHydA1 and CpI were produced adapting the method by Kuchenreuther.37 Briefly, electrocompetent E. coli BL21(DE3)ΔiscR cells were transformed with the respective expression plasmid.38 After aerobic cultivation at 37°C, cells were subjected to anaerobic conditions and protein expression was induced by adding Isopropyl βD-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 M. The expression cultures were further cultivated over night at room temperature (RT) under constant stirring (150 rpm) in an anaerobic tent (Coy Labs) under a nitrogen/hydrogen atmosphere (99:1). All solutions and buffers were supplemented with 2 mM sodium dithionite (NaDT). The strep-tagged protein variants were affinity purified with Strep-Tactin Superflow high capacity cartridges (IBA Lifesciences) according to manufacturer’s instructions. The purity of hydrogenase was monitored by SDS-PAGE and protein concentration was determined via Bradford assay (Bio-Rad) using bovine serum albumin as standard (Biolabs). Protein samples were concentrated to 1-2 mM in 100 mM Tris/HCl, pH 8.0 and stored at −80°C until further use. Preparation of (2FeH) Mimics. (2FeH) mimics were synthesized as described earlier.39 Crystalline compounds were dissolved in 100 mM potassium phosphate buffer (KPI), pH 6.8, and stored at −80 °C. In vitro Maturation of [FeFe]-Hydrogenases. Recombinant holoproteins of CrHydA1 harboring either the native azadithiole (adt)-derivative or the inactive synthetic cofactor [Fe2(pdt)(CO)4(CN)2]2− (pdt2− = propanedithiolate) was yielded upon in vitro maturation which was executed as described earlier with minor modifications.40 In this, the incubation time of apo

CrHydA1 and CpI with the adt-complex was held for 1 h at 4°C whereas the incubation with the pdt-complex was performed for 4h at room temperature (RT). Enzyme Activity Assays. In vitro hydrogen evolution activity was measured from 8 mL vessels (Suba) containing 400 ng enzyme, 10 mM methyl viologen (Sigma Aldrich) as electron mediator and 100 mM NaDT as sacrificial electron donor in a total volume of 2 mL 100 mM potassium phosphate buffer (KPI) (pH 6.8), by analyzing 400 µL head space via gas chromatography (Shimadzu) after purging with Argon (5 min) and sample incubation for 20 min in a shaking water bath (100 rpm) at 37°C. H2 uptake activity was followed spectrophotometrically by monitoring the reduction of benzyl viologen (BV) (Sigma Aldrich) at 600 nm under RT and 1 bar H2 in a reaction mixture containing 10 ng of protein in KPI-buffer (pH 6.8) and 10 mM BV (molar extinction coefficient ε = 10 mM⁻¹ cm⁻¹). Protein Film Electrochemistry. Electrochemical measurements were carried out using a gas tight three electrode electrochemical cell. The cell was water jacketed to provide temperature control during the experiments. A saturated calomel electrode (SCE) (Radiometer Analytical, France) was used as reference electrode, placed in a side-arm to keep it at room temperature, connected to the main cell compartment by a Luggin capillary. A Pt wire was used as counter electrode. Pyrolytic graphite “edge” (Momentive, USA) was used to build rotating disk electrodes (0.03 cm2 area) for protein adsorption. Potentials in this article are converted to standard hydrogen electrode (SHE) using the correction ESHE = ESCE + 241 mV at 298 K. The electrochemical cell contained a buffer mixture of each 15 mM MES, HEPES, TAPS, CHES and sodium acetate, as well as 0.1 M NaCl. CrHydA1 was adsorbed by drop casting a freshly polished electrode with 3 µL of protein solution (8 µM) for 5 minutes. Then the electrode was rinsed and transferred to the electrochemical cell. A VersaStat 4 400 (Ametek, USA) was used as a potentiostat. FTIR Spectroscopy. FTIR spectra were recorded using a Bruker IFS 66v/S FTIR spectrometer equipped with a nitrogen cooled Bruker mercury cadmium telluride (MCT) detector. Spectra were collected in the transmission mode at 15°C in a double sided, forward-backward mode with 1000 scans, and a resolution of 2 cm-1, an aperture setting of 1.5 mm and scan velocity of 20 kHz. EPR Spectroscopy. X-band EPR spectra were recorded on a Bruker ELEXSYS E580 pulse X-band spectrometer. The cryogenic temperatures were maintained by an Oxford CF935 Helium flow cryostat. Q-band EPR spectra were recorded on a Bruker ELEXSYS E580 pulse Qband spectrometer. Cryogenic sample temperature was controlled by a custom made closed cycle cryostat from Cryogenic Ltd. Davies 13C ENDOR spectra at Q-band were obtained using a home-built EPR/ENDOR resonator and a 300W ENI RF amplifier.41

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Figure 2: Selected FTIR spectra of 1-3 mM CrHydA1 (as isolated) in 100 mM Tris/HCl buffer pH 8.0, 2 mM NaDT measured at 15°C. The left panel shows spectra obtained for protein variants with mutations next to CNp and the right panel shows spectra for protein variants with mutations next to CNd. Signal positions for most prominent redox states are indicated at the top. Shifted peak signals can be monitored by dashed lines. A modeled structure of the (2FeH) site and the surrounding polypeptide framework from CrHydA1 (PDB ID: 3LX4) is shown on the right upper corner. Exchanged amino acid positions are highlighted by a black carbon backbone. Resulting interactions with the CO and CN ligands are indicated by dashed lines. Grey/Black: Carbon; Blue: Nitrogen; red: Oxygen, Yellow: Sulfur; Orange: Iron

3. RESULTS AND DISCUSSION Identification of Active Protein Variants. Protein variants targeting the secondary ligand sphere of CNp and CNd were selected from saturation mutagenesis libraries for retained enzyme activity based on a previously published colorimetric plate screening assay (for a detailed description of this procedure see Fig. S1).42 We identified variant S232A of CpI and in parallel the corresponding variant A94S in CrHydA1 in addition to A92S to be the most promising candidates to examine the influence of H-bond partners in the CNp environment of both [FeFe]-hydrogenases. The conservative CrHydA1 substitutions S193C and E231D were chosen to examine the influence of modifying the more sensitive environment of CNd. Kinetic Characterization of CrHydA1 Protein Variants. For all purified enzyme variants H2 production and H2 oxidation activities were determined in solution. All variants with mutations within the CNd ligand sphere exhibited strongly decreased H2 production ac-

tivities (ranging from 27% for E231D to merely 0.3% for S193A). The same trend can be observed for variants with double exchanges in the CNP ligand sphere (see table S1). This can be explained by a decreased incorporation of the (2FeH) site during in vitro maturation as the low relative signal strength of the diatomic (2FeH) ligand sphere in the FTIR suggests (Figure S5). H2 production at or exceeding WT level could only be observed for protein variants A92S and A94S which were fully maturated. The A92S/A94S double mutant showed 50% remaining activity which could be explained by a decreased H-cluster occupancy by 50% compared to WT. Consequently, the three variants A92S, A94S and A92S/A94S were chosen for an in depth analysis. FTIR Spectra of CrHydA1. Previous DFT calculations of the H-cluster showed that each of two FTIR signals in the 2100-2020 cm-1 region can be assigned to isolated stretching modes of one of the two CN- ligands.34 This suggests that exchanges in the close environment of individual CN- ligands should mainly affect only one of

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the two state specific signals, thus allowing a clear band assignment. However, CN- signal positions featuring a linewidth of 5-8 cm-1 are very similar in most redox states and are hardly affected by changes in oxidation states of the di-iron center.27 Additionally, “as isolated” (2 mM NaDT) [FeFe]-hydrogenase samples usually feature mixed spectra which comprise multiple redox states, resulting in partially overlapping patterns.40 To analyze the exchange effects as precisely as possible it is therefore more convenient to obtain a homogeneous spectrum from a single redox state, preferably from the super reduced state (Hsred) which lacks a bridging CO ligand. This enables a better CN- signal separation by 40 cm-1 compared to 15 cm-1 in states like Hox or Hred.16, 31 While wild type CrHydA1 has a higher contribution of Hox, the as isolated samples of variants A92S, A94S and double exchange variant A92S/A94S exhibit higher fractions of reduced states, predominantly Hred and Hsred (see Fig. 2). Most striking, the FTIR spectra of variants A92S and A94S show a bathochromic shift for only one of the two CN- signals. In case of A92S, the corresponding CNsignal is shifted by -15 (Hsred) to -19 cm-1 (Hred); the same signal is shifted by -22 cm-1 (in both, Hred and Hsred) for A94S (see Fig. 2). For variant A92S/A94S these shifts are even more pronounced (-29 cm-1 in Hsred) indicating a nearly additive effect from both single amino acid exchanges. As expected for state Hred which contains a bridging CO ligand (µCO) similar to Hox, CrHydA1 variant A92S shows an additional bathochromic shift of -7 cm-1 for the vibrational signal of µCO. The residue of polypeptide position 92 is situated between CNp and µCO and its in silico exchange (AlaSer) in the structure model of maturated CrHydA1 clearly results in overlapping van der Waals (vdW) radii for the introduced hydroxyl group of serine relative to both ligands, CNp and µCO (Fig S6 b). As a result, the local structure might be packed more tightly causing increased molecular interactions. This hypothesis can be corroborated by FTIR-spectra of the same variants made in the COinhibited states Hox-CO and Hred-CO (Figure S3) in which the packing density around the CO and CN- ligands is already more increased compared to Hred due to the additional CO ligand. Again this effect is even more pronounced for variant A92S/A94S in which a splitting of the µCO signal is observed suggesting two distinct populations. Interestingly, also the vibrational signals for the proximal and distal CO ligands (in all redox states) are slightly hypsochromically shifted by 2-6 cm-1 for all three variants which can be explained by an increased packing density around the (2FeH) site resulting from the incorporation of larger amino acid side chains. Furthermore, wild type CrHydA1 and protein variants A92S and A94S were maturated with the biomimetic pdt-complex featuring a CH2 bridge head group instead of the secondary amine of the native adt-derivative. Maturation with this synthetic complex results in the

nearly inactive enzyme variant CrHydA1pdt.35 However, this CrHydA1 variant can be stabilized in pure redox states; either Hpdtred or Hpdtox (upon thionine treatment). The corresponding FTIR spectra (Figure S4) clearly demonstrate that for variants A92S and A94S only the high wavenumber CN- signal is consistently bathochromically shifted for all redox states. This advocates for a distinct structural effect induced by the additional hydroxyl group of serine. As both targeted amino acid positions are located in the close proximity of the CNP ligand, an interaction with CNd can be excluded. Consequently, the results strongly suggest that the higher wavenumber CN- signal has to be assigned to the stretching mode of the proximal CN- ligand. This assignment is complemented and supported by FTIR spectra obtained from protein variants S193C and E231D with exchanges close to the CNd ligand sphere. Just like variants A92S and A94S the FTIR spectrum of E231D shows a selective bathochromic shift in the CN- vibrational region but in contrast to those variants, the shift now concerns the signal at lower wavenumber (Figure 2). In comparison, the shift is not as significant (-5 cm-1) as the shifts determined for variants A92S and A94S. However, this is expected considering that in this case the targeted amino acid only indirectly affects the coordination of CNd via its salt bridge contact to the side chain of K228, a direct and essential H-bond partner of the CNd ligand (see Figure 1).19 Furthermore, the exchange of glutamate to aspartate is a conservative exchange which retains the interacting carboxylic group. A similar selective bathochromic shift of -3 cm-1 can be identified for S193C which in contrast to E231 is in direct contact to CNd. In S193C the exchange effect results from the chemical and spatial difference caused by the different electronegativity and atomic diameter (48 pm in case of O (Ser); 88 pm in case of S (Cys)) of the chalcogen in the otherwise corresponding amino acid residue. Consequently, in both cases the effect on the overall coordination sphere of CNd is not as pronounced as for the applied Ala/Ser exchanges near CNp where the substitutions determine the presence or absence of one of the H-bond partners. With this supportive data, both CN- vibrations can be assigned to the individual CN- ligands. Accordingly, the spectroscopic data obtained from all examined variants unanimously suggest that in case of CrHydA1, the higher frequency signal in the CN- vibrational region can be assigned to CNP while the lower frequency signal is attributed to CNd. FTIR Spectra of CpI. As already mentioned above, residue A94 in CrHydA1 corresponds to S232 in CpI. Therefore, CrHydA1 variant A94S should mimic the situation of wild type CpI. Consequently, CpI variant S232A which targets the environment of CNp at the homologous position was expected to reproduce the situation of wild type CrHydA1. Indeed a selective shift was observed in the CN- specific region of FTIR spectra from variant S232ACp as visible for both examined states

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Figure 3: Selected FTIR spectra of 0.5 mM CpI WT and variant S232A in 100 mM Tris/HCl buffer pH 8.0 measured at 15°C. Left: -1 FTIR spectra for the Hox state. Note that the small feature at 2015 cm in WT CpI originates from traces of the Hox-CO state. Right: FTIR spectra for the Hox-CO state. The shift of the low frequency CN band induced by the S232A mutation is indicated by the dashed lines.

Hox and Hox-CO (Figure 3). According to our expectations the signals were hypsochromically shifted by 8 to 16 cm-1 thus, mirroring the bathochromic shift of the counter exchange in A94S. Interestingly, in case of CpI, the low frequency CN--signal of 2070 cm-1 (2075 cm-1 for Hox-CO) was affected while the position of the high frequency peak at 2082 cm-1 (2090 cm-1 for Hox-CO) remained unaffected by the exchange. This indicates that CpI exhibits an inverse CN- peak assignment compared to CrHydA1. These findings correlate well with the spectra from the CrHydA1 variant A94S, suggesting that a serine in that position shifts the CN- band to lower wavenumbers. Additionally, the vibrational signals of the proximal and distal CO ligands are now bathochromically shifted by 2-3 cm-1 (hypsochromically shifted by 2-4 cm-1 for CrHydA1). Accordingly the addition of Hbond partners to CNp causes a general hypsochromic shift among the vibrational signals of terminal CO ligands while their removal leads to a corresponding bathochromic shift. Such small but undeniable frequency shifts which are even more pronounced in the double exchange variant of CrHydA1 (+5 cm-1) could be explained by a decrease (CrHydA1 variants)/increase (CpI variant S232A) in electron density among the (2FeH) subcluster being an immediate consequence of accepting/removing an H-bond donor contact. This highlights the influence of the protein environment around the (2FeH) subsite in the electronic structure of the Hcluster, which has to be taken into account for an artificial peptide design and also for DFT calculations. EPR-Spectroscopy. The presented FTIR-spectroscopy data suggest that upon introduction of larger amino acids with side chains capable to form H-bonds to one of the negatively charged CN- ligands, a bathochromic shift of the corresponding signal is observed.

Figure 4: Q-band EPR spectra of WT, A92S, and A94S pdt CrHydA1 [FeFe]-hydrogenase in the H ox state. The slight shifts in g-values for the two variants can be observed with respect to the WT-CrHydA1 principal g-values indicated by the dotted lines. The asterisk indicates the signal of a small fraction of unwanted signal.

EPR-spectroscopy is potentially capable to characterize mutational effects in the secondary ligand sphere by probing electron-nuclei interactions for unpaired electrons that are delocalized over the H-cluster when isotopes like 1/2H, 14/15N and 13C are introduced.16

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Figure 5: Voltammetry experiments and fittings (dashed black traces) for the WT (orange) as well as the different protein variants (A92S, red; A94S, green; A92S/A94s blue) at pH 5, 7 and 9. Experimental conditions: 20 mV/s, 25ºC, 2000 rpm electrode rotation speed, 1 bar H2.

The oxidized state Hox is best suited for EPR studies targeting CN- ligand interactions because a substantial amount of spin density is known to be present at the CN- ligands.35, 43 For EPR experiments, wild type CrHydA1pdt and protein variants A92Spdt and A94Spdt were stabilized in the pure oxidized state which is an excellent model system for the Hox state in the active enzyme. Slight shifts to lower g-values are observed in EPR spectra recorded for variants A92S and A94S, compared to wild type CrHydA1 (Figure 4). Larger shifts of canonical g-tensor positions are observed for variant A94S, i.e. 0.0043 (g2) and 0.0021 (g3). These small shifts are comparable to effects observed in organic radicals, e.g. semi-quinones, caused by changes of the hydrogen bonds upon exchanging aprotic nonpolar for protic polar solvents.44-46 It is thus reasonable to assign the observed g-tensor shifts to changes in the surrounding of the CN- ligands. In solution activity assays. When comparing the ratios for H2 evolution and uptake measured by in-solution assays, a clearly shifted catalytic bias can be observed for the CrHydA1 variants A92S and A94S suggesting an influence of individual exchanges on the redox features of the H-cluster. As opposed to WT CrHydA1, hydrogen production of variant A92S is not favored over oxidation, exhibiting a nearly 1:1 ratio. In contrast, variant A94S has a slightly increased bias to hydrogen production activity and additionally shows a strongly reduced H2 oxidation rate (3:1 ratio). Regarding the A92S/A94S double variant, the activities for both catalytic directions are likewise lowered to almost 50% compared to WT (table S1 and figure S2). Protein Film Electrochemistry. The activity of the variants was further investigated using protein film electrochemistry (PFE). The protein variants A92S, A94S and A92S/A94S adsorbed on graphite electrodes show comparable catalytic currents, with maximum values around 800 µA cm-2 at pH 9 for H2 oxidation for the A92S variant (Figure 5). The currents were normalized to the maximum H2 oxidation current to facilitate visual analysis of the catalytic bias (see Fig. S10).

To analyze the catalytic bias, cyclic voltammograms (CVs) were measured from the equilibrium potential to 200 mV of overpotential in the directions of H2 oxidation and H+ reduction at the same driving force. While at neutral pH, the variants A92S and A94S do not show significant differences compared to WT, a change in the catalytic bias can be observed at more extreme pH values. In this, A92S has a slightly increased H2 oxidation activity at pH 5 compared to the WT, while variant A94S is more biased towards H2 evolution. At pH 9, neither the WT nor any of the variants evolve H2, but variant A92S displays an almost two-fold increased oxidation current compared to WT, following the same trend observed at lower pH values. The double variant has a lower activity at all three measured pH values compared to WT as a result of the lower fraction of fully maturated holoenzyme (see above). In general, the effects of both exchanges are reflected in the double variant. For the protein film voltammetry measurements the change in the catalytic bias is not as pronounced as for the insolution measurements, however the general trend can be confirmed. Taken together, variant A92S displays enhanced H2 oxidation while the A94S variant tends to be biased towards H2 evolution relative to WT. Moreover, it is worth noting that in order to obtain comparable and reproducible results between WT and the different variants, the same batch of (2FeH) mimic has to be employed. Additionally, the catalytic bias is also affected by several other experimental conditions that have to be controlled carefully (i.e. electrode employed, affecting interfacial electron transfer as well as electrolyte volume, gas flow and pH, affecting the substrate concentration in the electrochemical cell). All these factors prevented a more accurate quantitative analysis. Changes in the catalytic bias at pH 5 and 7 for the three enzyme variants are listed in table 1 compared to wild type CrHydA1. Table 1: Ratio of H2 oxidation/evolution at pH 5 and 7 and ±200 mV overpotential, obtained from cyclic voltammetry measurements.

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pH 5

pH 7

WT

0.56±0.06

1.53±0.06

A92S

0.5±0.1

2.1±0.1

A94S

0.29±0.02

1.59±0.02

A92S/A94S

0.62±0.04

3.2±0.5

The amino acid substitutions A94S and A92S affect the CNP ligand sphere presumably by interacting with the local H-bond network. According to the FTIRspectroscopic bathochromic shift of the CNP signal, the exchanges weaken the bond energy between N and C of CNp causing a bond elongation. At the same time a general hypsochromic shift can be observed (excluding the µCO signal in A92S) suggesting an overall decreased electron density at the (2FeH) center. Additional information can be obtained from fitting the experimental data to a model described by Fourmond et al. for this hydrogenase adsorbed on a carbon electrode.47 Using QSoas, the experimental data fit well to an EECr process (“E” referring to a one-electron redox step, “C” to a non-redox chemical reaction and “r” to a reversible catalysis process).48 The data fit better to the model that considers a dispersion of k0 values, as it is common for a protein adsorbed on a PGE (k0 is the interfacial electron transfer constant at zero driving force).48-49 This fit gave slightly better results than the EECr(R) model, which considers interfacial electron transfer between the electrode and a redox group acting as a relay between the protein surface and the active site of the enzyme (Figure S11). The model provides the potentials for the redox transitions from the most oxidized state to an intermediate state (EO/I) and from the intermediate state to the most reduced state (EI/R) (See section 4 in SI for additional information). Although the nature of states O, I and R cannot directly be identified in this experiment, it is tempting to assign these states to Hox, Hred/HredH+ and HsredH+, respectively. In general, the values obtained from this analysis match quite well the reported values for the corresponding redox transitions using an FTIR spectro-electrochemical cell.31 In that work, it is proposed that the reduction of the active site takes place always at the (4FeH) site of the H-cluster at similar potential values (which may explain why the EECr(R) model offers as well a reasonable fitting, since in the end the [4Fe4S]-cluster acts as a relay) and that the protonation of the bridging amine at the (2FeH) moiety of the H-cluster triggers an intramolecular electron transfer between both subclusters from Hred to HredH+.50 This protonation event has a pKa-value of 7.2 which may explain the observed shift of the potentials obtained from our simulations. Recently, it has also been proposed that protonation of the (4FeH) coordinating cysteine residues may play an important role in catalysis.51 Such protonation events may also influence the potential of the observed redox transitions.

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In order to obtain information about the catalytic bias we need to compare the E2H+/H2 with the appropriate mid potential for the two electron transition from the most oxidized state to the most reduced one (EO/R), calculated as the average of the two individual transitions (Figure 6, bottom panel).47 The analysis of the potentials obtained for the different variants reveals very small shifts for these transitions in comparison to the WT. If the enzyme has to produce H2, EO/R must be lower than the thermodynamic proton reduction potential (E2H+/H2, represented by a dashed black line in Figure 6). This explains why the enzyme loses the H+ reduction capability at a pH>8. On the other hand, the EO/R has to be more positive than the E2H+/H2 for H2 oxidation in order to allow the active site to receive electrons from the H2 molecule. The deviation of these two potentials from the E2H+/H2 creates a small overpotential that can be observed in the voltammograms. The A92S variant shows increased EO/R values in comparison to the WT which suggests a higher redox potential. This would fit to the overall slightly reduced electron density of the (2FeH) center reflected by the hypsochromic shifts in the CO ligand vibration signals that accompanies these exchanges. In turn, this increases the driving force for the H2 oxidation reaction resulting in increased H2 oxidation currents. In agreement with these results, for the [FeFe]-hydrogenase CpII of C. pasteurianum which naturally

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Journal of the American Chemical Society tial shifts for the individual one electron transitions, which is very evident from the CV shape, in which the enzyme requires a small overpotential to start the catalytic reaction on both directions. The resulting more positive EO/R for this variant explains the shift in bias towards H2 oxidation compared to the WT at neutral and basic pH values.

4. CONCLUSION Understanding how the active site coordinating sphere affects the properties of a metalloenzyme is fundamental in enzymology. It has already been demonstrated that the strongly conserved secondary ligand sphere around the (2FeH) cluster plays a major role in preserving a stable cofactor coordination and geometry. For CrHydA1, H-bonding of the involved amino acids does not only stabilize both CN- groups in trans configuration, but facilitates the formation of the open coordination site at Fed which is essential for fast catalytic turnover.19, 22

Figure 6: (A)Plot of the EO/I and EI/R obtained from fitting the voltammograms to an EECr model considering the enzyme in three oxidation states with two redox transitions and a chemical step, either H2 uptake or release (as shown in the scheme). The background colors show the regions where the oxidized (red), one-electron reduced (blue) or completely reduced (green) states should prevail. (B) Plot of the two electron EO/R. Each point on both figures represents the average value and the standard deviation from at least three independent measurements. The thermodynamic proton reduction potential is plotted as a black dashed line. Experimental conditions: 25ºC, 1 bar H2.

harbors a serine at position S99 (corresponding to A92 in CrHydA1) this position among a few others was discussed to shift the catalytic bias towards H2 oxidation.52 This is even further supported by the fact that the [FeFe]-hydrogenease CpI (naturally harbours a serine at A94 in CrHydA1) is biased towards H2 production. Implementation of each exchange in CrHydA1 demonstrated that a tuning of the catalytic bias into one specific direction is fundamentally possible. On the other hand, variant A94S has the lowest EO/R at pH 5. This increases the driving force for the H2 evolution reaction and therefore shifts the bias towards H2 evolution. The A92S/A94S variant has the largest poten-

Our work extends the role of the secondary ligand sphere apart from only being an anchor for the (2FeH) moiety. We provide first experimental evidence that manipulation of the secondary ligand sphere indeed influences the electrochemical properties of the active site and accordingly, the catalytic performance. The data suggest an influence on the catalytic bias achieved by exchanges A92S and A94S in either catalytic direction, depending on the pH. Additionally, these specific manipulations for the first time allowed for a discrete assignment of the two CN- ligands for two different [FeFe]-hydrogenases. While a similar approach could be undertaken for other metalloproteins the prospect of redirecting the catalytic bias towards H2 production for [NiFe]-hydrogensaes (in most cases biased towards H2 oxidation) might be even more tempting. However regarding both, the influence of product inhibition and electron transfer via a chain of additional FeS clusters, the catalytic bias of [NiFe]hydrogenases may represent a more complex and multifactorial phenomenon. To date, most inorganic mimics of the H-cluster have failed to deliver high turnover H2 production rates at low overpotentials and ambient system conditions.53 These results shed new light on the role of the secondary coordination sphere and will further interlink the fields of chemistry and biology to pave the way for establishing biotechnological applications for this highly promising enzyme family.

ASSOCIATED CONTENT Supporting Information. Further FTIR and EPR spectra are listed in the supporting information. This part also includes a detailed kinetic characterization of the protein variants from in-solution assays. This material is available free of charge via the Internet at http://pubs.acs.org.

9



AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Acknowledgements We would like to thank Birgit Nöring for her technical assistance in electrochemical measurements. AAV, OR, EJR and WL acknowledge funding from the Max Planck Society. This work was supported by the Cluster of Excellence RESOLV (EXC1069) funded by the Deutsche Forschungsgemeinschaft (DFG) for financial support. M.W. and T.H. further acknowledge financial support from the Volkswagen Foundation (LigH2t). Also, we like to thank the “Studienstiftung des Deutschen Volkes“ for financial support.

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Interplay between CNâ Ligands and the Secondary Coordination

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