X‐ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases

Abstract [NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational‐spectroscopic insights into the unexplored structure of the H2‐binding [NiFe] intermediate. Using an F420‐reducing [NiFe]‐hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit‐bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio‐inspired H2‐conversion catalysts.


Introduction
Mapping out strategies for future energy storage and conversion represents one of the central challenges of the 21 st century.M olecular hydrogen (H 2 )i sa ni deally clean fuel whose combustion releases large amounts of free energy but no greenhouse gases.T oe xploit it to its full potential, however, we require efficient and sustainable approaches for catalytic H 2 cleavage and formation.
[NiFe] hydrogenases are valuable model enzymes that catalyze H 2 conversion at rates comparable to platinum electrodes by using ah eterobimetallic active site containing cheap and earth-abundant base metals only. [1] Their rational utilization as biotechnological targets or blueprints for bio-inspired catalysts,h owever, requires at horough understanding of the structural and mechanistic determinants of their reactivity.Here,weemploy ab idirectional F 420 -reducing [NiFe] hydrogenase from the archaeon Methanosarcina barkeri MS (MbFRH) as au nique model system to yield structural and spectroscopic insights into as carcely explored reaction intermediate that is the initial target for H 2 binding to the active site of the enzyme. Thes tructure of this central catalytic species is analyzed in detail to explore its relevance for the mechanism and performance of [NiFe] hydrogenases.

Results and Discussion
Thec rystal structure of MbFRH was refined using reflections up to d min = 1.84 (Supporting Information, Table S1). Each asymmetric unit contains three subunits, FRH-A, FRH-B,a nd FRH-G,a nd at otal of four [4Fe4S] clusters as well as af lavin adenine dinucleotide (FAD) cofactor and the [NiFe] active site (detailed below). TheFAD and the heterobimetallic [NiFe] center enable the redox conversion of the two substrates,c oenzyme F 420 and H 2 , respectively,w hile the [4Fe4S] clusters mediate intramolecular electron transfer (ET) between the two reaction sites ( Figures 1A and S1). These features are shared with the related [NiFe] hydrogenase from Methanothermobacter marburgensis (Mm), and both enzymes exhibit ad odecameric overall architecture in as pherical shape ( Figure S1). [2] Compared to the latter enzyme,h owever, MbFRH contains two additional cofactors that are both ET-accessible and solventexposed:a[2Fe2S] cluster bridging two FRH-G subunits ( Figures 1A and S2) and am ononuclear Fe site in FRH-A ( Figures 1A and S3).
Based on element-specific anomalous scattering, the single Fe ion has also been assigned in [NiFeSe] hydrogenases, [3] while Ca 2+ [4] or Mg 2+ [5] ions are typically found at the same position in other [NiFe] hydrogenases (Table S2) In the following,wewill focus on the [NiFe] active site of MbFRH, which exhibits the consensus structural properties observed for other [NiFe] hydrogenases. [6] Specifically,t his heterobimetallic cofactor features two metal ions,Niand Fe, that are coordinated by four strictly conserved cysteinate (Cys) residues and three Fe-bound diatomic ligands.Based on infrared (IR) spectroscopic analyses,t he latter constituents are generally assigned to one CO and two CN À ligands. [7] Consistently,o ne CO and two CN stretching bands can be observed in IR spectra of MbFRH crystals,w hich confirms the presence of as tandard set of inorganic ligands (Figure 2A,b lack trace). Since these stretching vibrations are highly sensitive towards structural and electronic changes at the [NiFe] center, [8] theobservation of asingle set of three IR absorption bands in the relevant spectral range also demonstrates that the active site resides in ah omogenous redoxstructural state.Incontrast to crystal structures of many other [NiFe] hydrogenases,n oe lectron density can be detected in the third bridging position between the two metals for MbFRH ( Figure 2B). [6a-c] This excludes the presence of oxygen-containing ligands that would be indicative of inactive enzyme residing, for example,inthe Ni u -A or Ni r -B states. [6a,9] Notably,avacant third bridging site between the two metals has long been assumed to be ak ey feature of Ni a -S (also termed Ni-SIa), the H 2 -binding intermediate of [NiFe] hydrogenases. [6a,10] This assumption has been recently supported by spectroscopic analyses, [11] but detailed structural insights into this central catalytic intermediate have been so far unavailable.Inthe following,wewill shed light on these catalytic key aspects by using ajoint approach of crystallographic analysis and vibrational spectroscopy.
While the presence or absence of bridging oxygen ligands can be clearly established from crystal-structure analyses,H 2derived (hydride) ligands are only observable in sub-atomicresolution structures, [12] and available crystal structures lacking detectable bridging ligands most likely represent hydride species. [9e, 13] Since crystals of MbFRH have been prepared in am ildly reducing atmosphere containing up to 5% H 2 ,t he derived structural data could reflect Ni a -S or ah ydride species.T hus,b efore analyzing the active-site structure of MbFRH in more detail, we studied the underlying crystals using different vibrational-spectroscopic techniques to firmly identify the [NiFe] redox-structural state.
IR spectra of MbFRH crystals reveal CO and CN stretching bands at 1945 and 2065/2080 cm À1 ,r espectively, which resemble the IR fingerprint of the Ni a -S state of several [NiFe] hydrogenases ( Figure 2A). [6a] In particular, these vibrational frequencies are close to those observed for the as-isolated regulatory hydrogenase from Ralstonia eutropha (ReRH), which is aspectroscopically valuable yet structurally unexplored reference system for the Ni a -S state. [11] Based on this finding, contributions from the Ni a -C hydride intermediate and its photo-inducible congener,Ni a -L, can be excluded since these catalytic species would give rise to higher and lower CO stretching frequencies,r espectively. [6a] Thef ully reduced Ni a -SR state,h owever,c annot be excluded on the basis of IR-spectroscopic data alone since its dominating subspecies exhibits an IR fingerprint similar to that of Ni a -S in several [NiFe] hydrogenases. [6a] Thus,w en ext recorded resonance Raman (RR) spectra of MbFRH crystals to probe Fe À CO and Fe À CN metal-ligand vibrations as structural markers of the [NiFe] active site ( Figure 2C). [11b,14] In these measurements,N i a -SR would be photo-converted to Ni a -L, while Ni a -S would remain unaffected in terms of structural and electronic properties. [11b,14] Again, the obtained vibrational signature is very similar to that of the Ni a -S state as observed for ReRH and am embrane-bound hydrogenase from the same organism (ReMBH). [11b,14b] In particular, as tructurally sensitive vibrational mode with considerable FeÀCO stretching character can be detected at 596-598 cm À1 , which excludes contributions from Ni a -SR, since its photo- product Ni a -L would give rise to an Fe À CO stretching frequency above 600 cm À1 . [11b,14a] Moreover,t he intensity of the RR spectrum was found to increase with the excitation wavelength of the Raman probe laser, which is in line with previous observations for Ni a -S [11b,14] and contrary to expectations for reduced [NiFe] hydrogenases. [11b] Finally,w ea lso explored the effect of treating MbFRH crystals with CO,atypical inhibitor of [NiFe] hydrogenases. [6a, 15] We performed these experiments to check for interaction of CO with the [NiFe] active site,w hich is expected for Ni a -S (yielding Ni-SCO) but not for Ni a -C or Ni a -SR. [15d] Binding of extrinsic CO to the terminal vacant coordination site at the Ni ion of MbFRH is evident from the electron density at this position ( Figures 2F and S6) and ah igh-frequencyC Os tretching band at 2048 cm À1 in the corresponding IR spectrum ( Figure 2E), as also observed for other [NiFe] hydrogenases in the Ni-SCO state. [15a,c,d] Both structural and spectroscopic data show that this inhibited species accumulated to at least 50 %( Figure S6), while the remainder can be assigned to the Ni a -S parent state.Intotal, the above experiments show that the crystal structure of untreated MbFRH reflects ah omogenous Ni a -S state,a llowing adetailed analysis of this H 2 -binding intermediate.
Structural and electronic properties of Ni a -S have been proposed to be essential for efficient H 2 binding and hydride formation in [NiFe] hydrogenases. [11b,16] In the following,w e will revisit these proposals to evaluate their validity based on the crystal structure.While there is wide agreement regarding the overall catalytic mechanism of [NiFe] hydrogenases, details about the central steps of H 2 binding and activation are,sofar, elusive.Inparticular, the site of initial H 2 binding is not known, and either of the two metal ions may be involved in the formation of a(side-on) H 2 s-bond complex from Ni a -S. Experimental data on this first catalytic step are not yet available,but recent theoretical studies favor the Ni ion as the initial site of H 2 binding. [16][17] According to these studies,the coordination geometry of this metal ion represents ak ey to the energetically favorable interaction of H 2 with the [NiFe] Figure 2. A) IR spectra of an MbFRH single crystal (at 80 K, black) and ap rotein solution of ReRH (10 8 8C, gray). Spectra were normalized with respect to the CO stretching-band intensity.B)Crystal structure of the [NiFe] active site, exhibiting ad istorted seesaw coordination geometryo f the Ni(Cys) 4 moiety and avacant coordination site between the Ni and Fe ions. The 2 F obs ÀF calc electron density map after full refinementis shown as agray mesh (1.8 s). C) RR spectrum of an MbFRH single crystal (black) compared to that of ap rotein solution of ReRH.
[11b] RR spectra were recorded at 80 Ku sing 568-nm laser excitationa nd normalizedw ith respect to the most intense signal at 551/553 cm À1 .D)[NiFe] active-site crystal structure. Selected interatomicdistances are given in .E)IRabsorbancespectrum of an MbFRH single crystal, recorded at 80 K. Bands corresponding to the intrinsic, Fe-bound diatomic ligands and the extrinsic, Ni-bound CO of the Ni-SCO redox-structural state are highlighted in brown and orange, respectively.F)X-ray structure of the CO-inhibited [NiFe] active site. The 2 F obs ÀF calc electron density map after full refinement (1 s)a nd the residual F obs ÀF calc map (5.5 s)b efore CO-modeling are shown as agray mesh and green surface, respectively.Selected atoms and amino acid residues are shown as spheres (Ni in green, Fe in gray (B) and orange (D and F), Sinyellow) and sticks, respectively. active site.S pecifically,apeculiar seesaw-shaped geometry with trans S À Ni À Sa ngles approaching 1208 8 and 1808 8 was postulated to be mandatory for thermodynamically favorable binding of H 2 to Ni a -S. [16] In line with this proposal, the [NiFe] active site of MbFRH exhibits trans SÀNiÀSa ngles of 1078 8 and 1718 8,t hereby structurally confirming this unusual geometry of the Ni a -S state ( Figure 2B). This finding also agrees with previous RR studies on ReRH, [11b] highlighting the merit of combining crystallographic,s pectroscopic,a nd theoretical methods.
Notably,f our-coordinate Ni II sites,a sf ound in Ni a -S, typically exhibit (distorted) square-planar or tetrahedral coordination geometries.T his indicates that the atypical seesaw geometry of this [NiFe] intermediate is dictated by the four-cysteinate coordination pattern and the protein matrix of [NiFe] hydrogenases. [16,18] Remarkably, trans S À Ni À Sa ngles in the Ni a -S state of MbFRH resemble those found in computationally optimized Ni a -S geometries of previous theoretical studies (1248 8 and 1518 8), but comparison with the underlying crystal-structure-derived starting geometries (1098 8 and 1668 8)y ields an even better agreement. [16] This indicates that the magnitude and relevance of the structural constraints imposed by the protein matrix is even more pronounced than previously anticipated. Further evidence for the relevance of structural constraints comes from comparing the experimental NiÀFe distance of Ni a -S to values obtained in theoretical studies.C alculations on small-to medium-size models typically report Ni À Fe distances of up to 3.3 , [6a, 11b, 19] while models including larger parts of the protein matrix yield smaller values, [19] close to those we observe in the crystal structure (2.7 ;F igure 2D). This indicates that the protein matrix compresses the NiÀFe distance to values close to those observed for other catalytic intermediates,i ncluding hydride species,Ni a -C and Ni a -SR (2.6 ), [12] and presumed metal-H 2 adducts (2.6-2.8 ). [16,17b] This effect likely minimizes structural reorganization during H 2 turnover, thereby adding to the remarkable catalytic efficiency of [NiFe] hydrogenases. [11b] Additionally,ashort NiÀFe distance may also be relevant for metal-metal bond formation, as previously proposed for catalytic intermediates of [NiFe] hydrogenases. [20] Remarkably,N i À Fe distances obtained from large computational models only reproduce the experimental value observed for MbFRH if al ow-spin (S = 0) configuration of the [NiFe] active site is assumed in the calculations, [16,19] supporting asinglet ground state of Ni a -S in MbFRH.
To further explore the initial interaction of MbFRH with H 2 ,w ea lso investigated possible intramolecular H 2 transfer pathways.T othis end, MbFRH crystals were derivatized with xenon to explore hydrophobic gas channels connecting the exterior of the enzyme with the [NiFe] active site (Figures 3  and S7 A). These experiments revealed as econd H 2 -transfer channel that has not been observed in similar studies on other [NiFe] hydrogenases. [21] Surprisingly,t he unrelated [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough features as imilar hydrophobic tunnel ( Figure S7B), indicating analogous developments in certain hydrogenases that operate bidirectionally in vivo. [22] While this narrow channel appears obstructed in crystal structures of other [NiFe] hydrogenases (Figure S7 B), [21a,b] dynamically enhanced H 2 transfer via this route remains as af ar-reaching possibility and at arget for future studies.

Conclusion
In the current account we have experimentally explored the unique structural properties of the H 2 -binding catalytic intermediate of [NiFe] hydrogenases.Besides revealing additional electron and H 2 pathways,t he spectroscopically validated crystal structure of ap ure Ni a -S state unraveled two key determinants of efficient H 2 cycling:apeculiar seesaw-shaped coordination geometry of the Ni ion and as hort Ni À Fe distance that is indicative of al ow-spin electronic ground state.B oth structural observations contradict expectations for unconstrained low-molecular-weight transition-metal compounds,t hereby illustrating the central role of chelating ligand scaffolds and outer coordination layers in biocatalytic H 2 cycling.T hese findings expand our understanding of [NiFe] hydrogenases and, thus,p rovide valuable guidance for the future design of bio-inspired catalysts for H 2 -based energy-conversion approaches.