Mechanistic Principles of Hydrogen Evolution in the Membrane-Bound Hydrogenase

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.


■ INTRODUCTION
−6 Mbh powers the ferredoxin-driven (E m = ca.−480 mV at pH = 7) reduction of protons to H 2 (E m = −420 mV at pH = 7), 1,7−9 which is catalyzed by its [NiFe] active site.Mbh transduces the free energy (−60 mV/electron; −120 mV = −2.7 kcal mol −1 for the 2 H + + 2e − → H 2 reaction) into proton pumping (H + ) and sodium (Na + )/proton (H + ) exchange, generating a sodium motive force (smf) across the biological membrane that powers the Na + -dependent ATP synthesis of P. furiosus. 10Mbh is a 300 kDa transmembrane protein comprising 14 subunits, with the hydrophilic domain harboring the [NiFe] active site (in MbhL) and three iron− sulfur (FeS) clusters (in MbhN, MbhJ), responsible for the electron transfer, whereas the membrane domain catalyzes Na + /H + exchange (MbhA-G) and proton pumping (MbhM/ MbhH) (Figure 1).It is closely related to the respiratory Complex I (NADH:ubiquinone oxidoreductase), 3,11 a large (0.5−1 MDa) redox-driven proton pump that drives electron transport and oxidative phosphorylation in aerobic respiratory chains.Mbh is thus a key enzyme for understanding not only the evolution of complex bioenergetic machineries but also how H 2 gas production powers the generation of an ion motive force across a biological membrane.Despite significant molecular understanding of soluble [NiFe] hydrogenases, 12−15 their mechanistic principles remain puzzling and poorly understood.
For Mbh, the electrons required for the proton reduction are delivered by ferredoxin (Fd), 16 a small (7.5 kDa) soluble electron transfer protein that docks to the MbhN subunit 2 (Figure 1A).Fd is a one-electron donor that stepwise transfers the electrons via the three iron−sulfur clusters [4Fe-4S] located in MbhN and MbhJ to the [NiFe] center.The [NiFe] cluster (located in MbhL, Figure 1A) coordinates Cys71 L , Cys377 L , Cys68 L , and Cys374 L , and in addition, the Fe center binds two CN − ligands and one CO ligand (Figure 1B).Interestingly, the [NiFe] active site has evolved into the Qreduction site of the modern respiratory Complex I, with a striking structural resemblance together with key modular adaptations 3,11,17−19 (Figure 1D).−28 In the experimentally characterized Ni− SI a 20 and Ni−R 29 states, the center remains in the Ni II LS form, whereas the nickel is paramagnetic (S = 1/2) in the Ni− L 30−32 and Ni−C 33 states, with Ni I and Ni III , respectively.Recently, also a high-valent LS Ni IV (S = 0) state was assigned based on Fourier-transform infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) spectroscopic studies in a NAD + -reducing [NiFe] hydrogenase. 22In this regard, it was proposed that the hexa-coordinated Ni IV can protect the active site from O 2 -induced oxidative damage.However, despite the involvement of multiple spin and oxidation states, the role of spin crossover in H 2 catalysis is not well understood. 26,27,34n addition to the unclear molecular principles underlying H 2 formation, the pathways used for proton delivery to the active site in Mbh also remain debated.The protons required for the H 2 evolution in the soluble [NiFe] hydrogenases are most likely transferred via a pathway involving Cys546 and Glu34 (D. vulgaris Miyazaki F., hereafter DvMF; 35,36 Figure S1) that connect via a hydrogen-bonding network to the bulk solvent. 15,24,36,37Moreover, another pathway, via His82 (His75 L in Mbh, His88 in DvMF, also known as "histidine pathway"), was described in O 2 -tolerant membrane-bound [NiFe] hydrogenase from Ralstonia eutropha. 38,39In Mbh, four possible proton pathways lead from conserved Glu21 L (see below) to the [NiFe] center via water networks at the interface between MbhL/M, MbhL/I/J, MbhL/N/J, and MbhM/cleft. 5 These putative pathways have also resemblance to proton pathways leading to the Q oxidoreduction site in Complex I, 40,41 although their function remains unclear.
In contrast to soluble hydrogenases, the H 2 catalysis is functionally coupled to the ion pumping activity in the membrane domain of Mbh. 1 While the overall metal coordination has remained conserved relative to the soluble [NiFe] hydrogenases, Mbh has certain key structural differ- ences around the [NiFe] center that could mediate the coupling effects.One such modular adaptation is established around the conserved β1-β2 loop structure (linker region Ile10 L -Lys24 L ) of the MbhL subunit, which harbors Glu21 L , a residue that could shuttle protons to the active site 5,42 (see Figure 1D).The same loop undergoes conformational changes in respiratory Complex I 43−45 and could thus trigger the proton transport activity in the membrane domain of Mbh.The distance between Glu21 L and the Ni-coordinating Cys374 L is rather large (4.8 Å) for a direct proton transfer in the cryo-EM structure of Mbh (PDB ID: 6CFW 1 ), and the carboxylate side chain is poorly resolved.In this regard, recent studies 5 suggested that Glu21 L could undergo a conformational change, similar to the proton shuttling Glu-242 in cytochrome c oxidase, 46 and support proton transfer to the active site.Interestingly, similar structural motifs around the active site are also present in the membrane-bound formate hydrogenlyase (FHL), 6 which also couples H 2 formation with ion pumping.In FHL, the distance between Cys531 E and Glu193 E is around 4 Å in the cryo-EM structure (PDB ID: 7Z0S) and 7 Å in the aerobic preparation (PDB ID: 7Z0T), 6 whereas the homologous Glu193 E of the soluble [NiFe] hydrogenases is located around 3.4 Å from Cys546 in the highresolution crystal structure, 36 and may also provide the protons for catalysis.
Here, we probe the energetics of the catalytic cycle responsible for the elusive proton reduction and H 2 formation in Mbh by combining large-scale quantum chemical models with density functional theory (DFT), correlated ab initio wave function-based methods, and classical atomistic molecular dynamics (MD) simulations.Based on our multiscale approach, we identify the role of conserved residues in catalysis and how electric field effects modulate reaction barriers.Our work provides a molecular basis for understanding the link between H 2 catalysis and the ion transport activity across the membrane domain and highlighting key differences relative to the soluble [NiFe] hydrogenase, with implications for the evolution of the Complex I superfamily.Our work also provides new insight into the [NiFe] catalysis, including the involvement of different spin states, structural assignments of Ni−R and Ni−L states, and the role of the Glu and His pathways during proton uptake that have remained much debated.

Proton Transfer Energetics in [NiFe] Catalysis.
To obtain insight into the redox-triggered proton transfer energetics in Mbh, we first constructed large quantum chemical density functional theory (DFT) models of the [NiFe] active site.The models included, in addition to the bimetallic core and its immediate ligands, all first and second sphere protein residues and water molecules obtained from our atomistic molecular dynamics (MD) simulations, leading to a molecular system with around 260 atoms (see Figure 1B and Methods).It should be noted that the water molecule between Cys374 L and Glu21 L is not resolved in the cryo-EM structure of Mbh.Additionally, it is absent in the structure of soluble hydrogenases, as Cys546 and Glu34 form a direct hydrogen bond. 36Based on results from our current detailed MD simulations (see Figure 4A−C) as well as our previous work, 5 we have positioned a water molecule between Cys374 L and Glu21 L , where it shows an extended residence time in the MD simulations.Based on the DFT models, we further optimized reaction pathways for proton transfer leading to H 2 formation along the four experimentally characterized redox states (Ni− SI a , Ni−L, Ni−C, and Ni−R) of the catalytic cycle (see Figure 1C).Our extensive benchmarking calculations relative to highresolution (0.89 Å) X-ray data 36 as well as ab initio wave function theory (RPA, DLPNO−CCSD(T 1 )) show that the employed DFT methodology provides an accurate description of the geometry, electronic structure, and reaction energetics of the [NiFe] site (Figures S1−S3, Tables S1−S4).
We first explored the structure of the [NiFe] in the Ni−SI a state, with the metal core modeled in the Fe II /Ni II configuration, with the nickel in the singlet (S = 0, LS) or triplet (S = 1, HS) state (Figure 2A).To obtain insight into the proton transfer energetics, we permutated the proton on the possible protonatable residues around the active site (Glu21 L , Cys374 L , Cys377 L , Cys68 L , and His75 L ) and optimized reaction pathways for the proton transfer reaction between the different sites (Figures S5−S7 and Tables S5−S7).Overall, the optimized geometries (Figures 2B, S5, S6) suggest that the coordination environment around Ni II in the low-and highspin forms is almost identical with "see-saw"-like coordination (Figure 2B); this could arise from the restricted movement of ligands due to structural constraints imposed by the protein scaffold.However, we observed subtle differences in the metal−ligand bond lengths between the spin states (e.g., Ni− S C377 is 2.19 and 2.38 Å in 1B LS and 1B HS , respectively).See Table S6 for a detailed analysis).
The water-mediated proton transfer from Glu21 L to Cys374 L has a modest free energy barrier (ΔG ‡ = +12.7 kcal mol −1 in LS, ΔG ‡ = +8.8kcal mol −1 in HS), but protonation of Cys374 L is energetically unfavorable (ΔG = +6.5 kcal mol −1 in 1D LS ; + 5.3 kcal mol −1 in 1G HS ) (Figure 2A, see also Figure S7).When Glu21 L is protonated, both spin states are nearly isoenergetic (ΔΔG = 1.6 kcal mol −1 ), with a small overall preference for the HS form (Figure 2A).We find that the proton transfer from Glu21 L to Cys68 L (+15.6/+15.4kcal mol −1 for 1E LS /IE HS ) or from His75 L to Cys377 L is also energetically highly unfavorable (Figures S5, S6).The energetics compares well with our wave function-based ab initio calculations, suggesting that our hybrid density functional treatment captures the reaction energetics of the system within an overall accuracy of a few kcal mol −1 for the electronic effects (Tables S3, S4).Taken together, these calculations show that the overall proton transfer from Glu21 L or His75 L to the cysteine ligands or to the metal site in the Ni−SI a state is unfavorable (see also Table S6 for structural analysis).
One-electron reduction of Ni−SI a leads to the Ni−L state, with a Ni I /Fe II (S = 1/2) configuration, with the reduction strongly favoring the proton transfer toward the [NiFe] cluster (Figures 2D, S8, S9, Movie S1).In this regard, the proton transfer from Glu21 L (3A) to Cys374 L (3Y2) becomes strongly exergonic upon formation of the Ni−L state (ΔG = -7.0kcal mol −1 ) with a small reaction barrier (ΔG ‡ ∼ 2.0 kcal mol −1 , Figure 2D).The rotation of the protonated thiol of Cys374 L toward the [NiFe] site is nearly isoenergetic (see below, 3B, −6.5 kcal mol −1 ; ∼92°clockwise with respect to −SH group in 3Y2, see Figure 2D), with a two-center Ni−Fe metal−metal bond (Figure 2E, Tables S8, S9) forming at the core (cf.also ref. 31).3B and 3Y2 differ only in the rotated thiol group of Cys374 L , with both states featuring a short Ni− Fe distance of 2.57 Å.The proton transfer to Cys68 L (3G1, −4.1 kcal mol −1 , Figure S8) is also exergonic, suggesting that the proton could populate different rotameric configurations of both Cys374 L and Cys68 L , consistent with the experimentally observed multiple forms of the Ni−L state. 47,48However, in contrast to the energetically favored Glu21 L -mediated pathway, we find that His75 L -mediated proton transfer to the cluster is strongly endergonic (Figure S8) in the Ni−L state.Taken together, our findings suggest that the initial proton transfer reaction energetically favors the glutamate pathway (but see below).
Subsequent oxidation of the Ni center forms a Ni III /Fe II (S = 1/2) configuration, known as the Ni−C state.This reduces the transferred proton to a hydride (H − ) ion, mediated by a metalto-ligand electron transfer between the Ni and the H + (Figures 2D, S8, S9, Tables S8, S9).Protonation of the metal-core involves further rotation of the Cys-H bond, allowing the H − to coordinate to the bridging (μ-H − ) position (3B →3D, ΔG ‡ = +5.4kcal mol −1 with both the Ni III and Fe II S9, Movie S2).The established state closely resembles the experimentally characterized Ni−C form of the soluble [NiFe] hydrogenases. 49In contrast, we find that the formation of a terminal Ni−H S8, see also Table S9 for geometric analysis).The free energy profiles thus suggest that the conversion of Ni−L to Ni−C state is exergonic (3Y2 → 3D, ΔG = -8.7 and ΔG ‡ = +5.8kcal mol −1 ), and support the progression of the reaction toward the reduced metal-bridged (Ni−H − −Fe) hydride state (Figure 2D, E), but the additional protonation of the cysteine ligands is energetically unfavored in this state (Figures S10, S11), preventing further proton uptake in the Ni−C state (see below).
Electric Field-Induced Proton Transfer Reactions.To probe the molecular basis for the redox-triggered change in reaction barriers and the thermodynamic driving force for proton transfer in the Ni−SI a → Ni−L transition, we quantified electric field (E) effects around the active site.Our calculations suggest that the formation of the reduced Ni− L state increases the electric field along the proton pathway leading from Glu21 L to Cys374 L by |ΔE f | = 0.6 V Å −1 relative to the oxidized Ni−SI a state, with the reduction resulting in an electric field vector that points along the proton pathway toward Cys374 L (Figure 2F, G), with a stronger field effect relative to other coordinating residues (Figure S26).To further test how the electric fields could modulate the reaction barriers, we applied an external field in the direction of the H 2 O → Cys374 L bond in the Ni−SI a state.Interestingly, this applied field similarly lowers the reaction barriers and reaction energy with a linear shift with increasing field strengths that closely resembles the effects observed upon formation of the Ni−L state.These findings suggest that redox-triggered electric field effects could drive the proton transfer toward the [NiFe] center (Figure 2C) by similar effects as observed for other energy-transducing systems, such as cytochrome c oxidase, 50 Complex I, 51 and Photosystem II 52 (cf.also ref. 51).
Mechanism of H 2 Formation in the Ni−R State.Our DFT calculations suggest that the further one-electron reduction of Ni−C leads to a Ni II /Fe II configuration, known as the Ni−R state, with energetically accessible LS and HS forms.To probe the thermodynamic and kinetic feasibility of transferring the second proton required for the H 2 formation in this state, we studied the energetics of proton transfer from both Glu21 L and His75 L toward the hydride bridging the [NiFe] core, and based on these, we explored the energetics of forming H 2 within the [NiFe] core.
We find that the proton transfer from Glu21 L (4A LS/HS ) to Cys374 L (5E2 LS/HS ) becomes strongly downhill in the Ni−R state (ΔG = −14.7/−12.0kcal mol −1 in LS/HS, Figure 3), whereas the proton transfer to Cys68 L is endergonic (by ca.ΔG = +5.2/+3.2kcal mol −1 , see Figures S12−S16, Tables S10−S12).The protonation of Cys374 L has a small reaction barrier (4A LS/HS → 5E2 LS/HS , ΔG ‡ = +7.7/+5.6 kcal mol −1 for LS/HS), which supports the fact that the reaction is kinetically feasible.Moreover, due to the small energy gap between the spin states, the proton transfer step could employ spin crossover between the LS and HS configurations, which in turn would reduce the reaction barriers along the HS pathway (Figure 3).It is to be noted that the reaction barrier for the LS pathway is significantly reduced when His75 L is protonated (see below).Similarly, as for the first proton transfer step (see above), we find that the forward progression of the reaction involves rotation of the thiol group of Cys374 L (5E2 LS/HS → 4B LS/HS , ΔG = +6.1/+6.0kcal mol −1 for LS/HS) toward the metal-bound hydride.From here, the H 2 formation takes place by the oxidative addition at the Ni II center, thereby forming a dihydride-bound Ni IV /Fe II species in the LS state (4B1 LS , Movie S3, 4B LS → 4B1 LS , ΔG ‡ = + 6.3 kcal mol −1 ), with an octahedral coordination around a putative Ni IV state.Subsequent reduction of the Ni IV to Ni II (4B1 LS → 4D LS , ΔG = +0.3,ΔG ‡ = +2.8kcal mol −1 ) couples to the formation of the H 2 in the Ni II form (4D LS , Ni−H 2 is 1.59 Å).In contrast, the H 2 formation along the HS surface is highly endergonic (4B HS → 4E HS , ΔG = +12.2,ΔG ‡ = +17.9kcal mol −1 , Figure 3  Involvement of the Histidine Pathway in H 2 Formation.To probe the possible role of His75 L in both H 2 formation and proton delivery, 38,39 we also optimized all putative states along the catalytic cycle, but with His75 L modeled in its protonated (HisH + ) form (Figures S17−S25, Tables S14−S21).For the Ni−R state, we find that the proton transfer from His75 L (5A LS ) to Cys377 L (5B LS ) is exergonic and has a small reaction barrier (5A LS → 5B LS , ΔG = -4.8,ΔG ‡ =+0.3 kcal mol −1 , Figure 3).Tilting of the Cys377 L (5C LS ) allows the −SH group to move away from the bridging position (5B LS → 5C LS , ΔG = −0.6 kcal mol −1 ) that leads to a subtle shift in the side chain of His75 L (Figure 3B).The proton transfer from Cys377 L (5C2 LS ) to Cys374 L (5E2 LS ) is exergonic (ΔG = −7.3kcal mol −1 ) with a small reaction barrier (ΔG ‡ = +6.2kcal mol −1 ) (Figures S12−S14), while the subsequent proton transfer from Cys374 L toward the hydride is also energetically feasible along the same mechanism as discussed above (5E2 LS → 4B LS → 4B1 LS → 4D LS ).Interestingly, the proton transfer along this pathway is exergonic in the LS configuration but endergonic in the HS state (5A HS → 5B HS , ΔG = +3.4kcal mol −1 and ΔG ‡ = +4.8kcal mol −1 , Figures 3, S14−S16).As for the Glu pathway, our analysis suggests that electric field effects could also play an important role in barrier modulation for the His pathway (Figure S26).
In addition to its possible role as a proton uptake pathway, we found that the protonation of His75 L modulates the proton transfer energetics along the Glu pathway.In this regard, the proximity of the positively charged HisH + next to the [NiFe] center leads to an increase in proton transfer barriers for  S22 and S23.
Glu21 L to Cys374 L in the Ni−SI a to Ni−L transition (Figures S17−S21), as well as an increase in the reaction barrier for hydride binding during the Ni−L to Ni−C transition (see also Figure S21).In contrast, the proton transfer barrier from Glu21 L to Cys374 L decreases by +5.4 kcal mol −1 in the LS Ni−R state (Figures S23−S25).The latter effect could arise from a directional electric field (ΔE f = 0.1 V Å −1 ) formed along the Glu21 L -Cys374 L pathway upon the protonation of His75 L (Figure S26).We find that the His75 L protonation favors the HS over the LS form in the Ni−SI a state (Figure S19), but does not affect the spin energetics in the other redox states (Figure S25).Taken together, these findings suggest that protonation of His75 L could act as a pH switch that modulates the reaction energetics, consistent with altered [NiFe] activity dependence on the pH and its possible functional "pH sensing". 32aken together, our findings demonstrate that the second proton transfer required for H 2 catalysis could occur via either the "Glu pathway" (in both LS and HS configurations) or the "His pathway" (in the LS configuration).The latter requires protonation of His75 L , suggesting that the His pathway could be active at low pH upon formation of the Ni−R state and regulate the activity of the [NiFe] center.
H 2 Release and Active-Site Recovery to Ni−SI a .Our DFT calculations suggest that the binding of H 2 in the Ni−R state is modulated by the spin state.In the LS form (4D LS ), H 2 prefers binding in a side-on, η 2 configuration to Ni II , whereas H 2 binds in a bridging position between Ni and Fe in the HS configuration (4E HS ), although the state is energetically highly unfavored (ΔG HS-LS = +10 kcal mol −1 , Figure 3A).However, dissociation of H 2 leads to crossing of the spin states (4D LS → 4D HS , ΔG = +0.3kcal mol −1 ), and results in a degenerate apo-Fe II Ni II state for both the HS and LS forms (ΔG = +0.2kcal mol −1 ).These findings suggest that H 2 formation and dissociation could involve spin crossover.We find that the H 2 dissociation is coupled with a rather large entropic (TΔS) effect of −9.3 kcal mol −1 that could be relevant for transducing the free energy for ion transport (Figure S13).Our calculations further show that the release of H 2 restores the Ni−SI a state.
Redox-Triggered Conformational Changes Trigger the Ion Transport Machinery.In order to probe the conformational dynamics coupled to catalysis, we performed atomistic molecular dynamics (MD) simulations of Mbh embedded in a membrane−water−ion environment to probe how the redox-coupled proton transfer reaction induces conformational and hydration changes (Tables S22, S23).In this regard, we derived atomistic force field parameters based on the quantum chemical models for the [NiFe] cluster along the key steps of the catalytic cycle which allowed us to study the coupling between the redox catalysis and the conformational dynamics. 5ur MD simulations suggest that reduction of the [NiFe] center changes the conformational dynamics of Glu21 L , and leads to an increase in the "flipped-in" conformation of the residue.This increases the occupancy of the proton transfermediating water molecule, bridging the Glu21 L − Cys374 L gap during Ni−SI a → Ni−L and Ni−C → Ni−R transitions (Figure 4). 5 Interestingly, the proton transfer from Glu21 L to Cys374 L induces a conformational change, which leads to dissociation of the water molecule and favors the outward flipping of the anionic Glu21 L (Figure 4A−C), which could prevent back transfer of the proton from Cys374 L to Glu21 L / bulk solvent but also provide a possible coupling element that triggers ion transport in the membrane domain of Mbh (see below).Recent cryo-EM structures of the FHL complex 6 also show a cavity between Cys374 L and Glu21 L in one structure (PDB ID: 7Z0T, with a distance of around 7 Å) that could occupy a similar proton transfer-mediating water molecule, but not in an anaerobic sample (PDB ID: 7Z0S, with a distance of around 4 Å).Moreover, this feature has not been reported for soluble [NiFe] hydrogenases (DvMF).However, we note that although Cys546 and Glu34 form a strong hydrogen-bonding interaction in DvMF, FTIR experiments 24 suggest that a "dangling" water molecule realigns upon changes in protonation states during the Ni−C → Ni−L transition.The outward flip of the protonated Glu21 L in our simulations of the Ni−C state further stresses the possible functional role of His75 L in providing a second proton for the H 2 formation (Figure 3B).Sequence and structure comparison with the Complex I superfamily (Figure S30) show that Glu21 L occupies the same position as His38 Nqo4 (T.thermophilus numbering), which is likely to function as a proton donor in the quinone reduction process in Complex I 40 (cf.−55 ).
We next analyzed how the redox and protonation changes in the active site could activate the ion transport activity across the membrane by probing conformational changes in the membrane domain and surrounding loop structures.In this regard, it was recently suggested 44 that the quinone reduction in Complex I couples to conformational changes in the surrounding loop regions that could trigger a π-to-α transition in the transmembrane helix (TM3 ND6 ), enabling proton transport in the membrane domain.Interestingly, many of these loop regions are also conserved in Mbh (Figures 4D−H S27, S28).In the Ni−C state, we observe an outward flip of the protonated Glu21 L and a contraction of the β1-β2 loop, which results in the dissociation of the Lys24 L -Asp379 L ion pair.This conformational change destabilizes an ion-paired network involving Lys40 L , Lys9 L , Glu49 I , and Asp50 I , similarly as in Complex I. 44 Interestingly, when the MD simulations are performed in other catalytic states (Ni−SI a , Ni−L, Ni−R), we observe stable interactions between the loop region of TM1−2 and β1-β2.Destabilization of the ion-paired network between the TM1−2 and the β1-β2 loop in the Ni−C state leads to a large-scale conformation change of the TM1−2 loop that propagates toward the membrane arm (Figure 4H), consistent with the high B-factors and blurred density of the TM1−2 loop observed in the cryo-EM data. 1 Despite common redox-driven electrostatic and conformational changes that resemble the coupling elements suggested for Complex I, we find that many of the charged residues in TM1−2 (Glu49 I , Glu52 I , Lys53 I ) are unique to Mbh.Moreover, the region is more hydrophobic in Complex I relative to MbhM−a feature that could have evolved to support binding of the nonpolar quinone substrate.

■ DISCUSSION
In this work, we have proposed a molecular mechanism of the ancient membrane-bound hydrogenase, which reduces protons to form H 2 gas and couples this redox chemistry to ion pumping across the archaeal membrane.To this end, we derived an electronic structure-level understanding of the H 2 formation at the [NiFe] active site and probed how the redoxdriven large-scale conformational changes in loops connect the electron transfer domain to the membrane module.

Journal of the American Chemical Society
Despite some catalytic similarities to soluble hydrogenases, 14,15,23 Mbh also shows notable differences in its proton transfer mechanisms (Figure 5A).Two distinct proton transfer pathways are identified in Mbh that are operational based on the [NiFe] redox state.Our results suggest that the first proton transfer (in the Ni−SI a → Ni−L transition) takes place via Glu21 L (Glu pathway), while the second proton (Ni−C →Ni−R transition) can occur either via Glu or the His (His75 L ) pathways.Remarkably, the reaction barrier for the proton transfer between Glu21 L and Cys374 L during the Ni−C → Ni−R transition is higher (7.7 kcal mol −1 ) than that in the Ni−SI a → Ni−L transition (2 kcal mol −1 ) (Figures 2D and  3A).However, the barrier decreases (by ca. 5 kcal mol −1 ) in the presence of a protonated His75 L due to electric field effects (Figure S25).These findings suggest a possible interplay between the two proton transfer pathways in facilitating the delivery of the second proton to the active site.Previous experiments on the soluble [NiFe] hydrogenase from Pyrococcus furiosus found large kinetic isotope effects (KIEs) of the Ni−SI a →Ni−C conversion (via Ni−L) in the range of 6−43. 38Given that Cys374 L and Glu21 L share a strong hydrogen bond, the high KIEs suggest that quantum nuclear effects could play an important role in the proton transfer reaction, in addition to the low reaction barrier of 2 kcal mol −1 (Figure 2D) that also supports a fast transition.
It is to be noted that the coordination environment around Ni and Fe in different catalytic states could also play a critical role in the activation of the two proton pathways.Specifically, in the Ni−L state, we observed a strong coordination between Ni and S (Cys377 L ), with a bond length of 2.30 Å, while in the Ni−R state, this bond length increased to 2.74 Å, while the Ni−S (Cys374 L ) bond distance remained nearly identical in  [1] one-electron reduction of the [NiFe] by the proximal FeS cluster, which induces a directed electric field toward Cys374 L that [2] leads to proton transfer from Glu21 L to Cys374 L .The "flipped-in" orientation of Glu21 L favors the proton transfer and formation of an ion-paired interaction between Lys24 L and Asp379 L .Bottom inset: Following the proton transfer step: [1] Glu21 L flips into its "outward" conformation, which [2] favors dissociation of the Lys24 L -Asp379 L ion pair.Subsequently, the β1-β2 loop undergoes a contraction [3], which in turn favors the dissociation of ion pairs between the TM1−2 and the β1-β2 loop [4].This cascade leads to conformational changes in the TM1−2 loop, which moves toward the membrane domain.The motion is supported by contacts between Lys53 I and Glu375 H . [6] These conformational changes also lead to the formation of ion pairs between TM1−2 (MbhI), PSST (MbhJ), and TM5−6 (MbhM) loops.The interaction of the TM1−2 loop with the MbhH subunit via Lys53 I -Glu375 H is shown, together with key residues along the putative proton pathway in MbhH.
both states.This suggests a weaker binding of Cys377 L to the Ni in the Ni−R state as compared to the Ni−L state, indicating that it could tune the pK a values of residues and possibly alter the ligand field environment to facilitate proton transfer along the His pathway during the Ni−C → Ni−R transition.The activity of the Glu pathway during Ni−C →Ni−R is likely to also depend on the conformation of the β1-β2 loop, which harbors Glu21 L , and could function as a coupling element during the redox-driven proton pumping.
In soluble hydrogenases, both protons could be transported via the Glu pathway. 15In this regard, Evans et al. 56 studied the role of Glu28 (Glu34 and Glu21 L in DvMF and Mbh, respectively) in the O 2 -tolerant [NiFe] Hyd1 from Escherichia coli during H 2 oxidation by using an E28Q mutant, which accumulated mainly the Ni−R and Ni−C states.These findings led to the conclusion that Glu28 is not essential for the proton transfer during the transition from Ni−R to Ni−C, while the residue was found to be critical in the subsequent Ni−C → Ni−SI a transition.These results are consistent with our work, in which we suggest that the first proton originates exclusively from the "Glu" pathway, while the second proton could be transported along either pathway.The two proton transfer pathways in Mbh thus show a unique resemblance to the proton transfer linked to Q reduction in Complex I, where both His38 Nqo4 (part of the β1-β2 loop) and Tyr87 Nqo4 function as the likely proton donors. 40e note that several structural models for the Ni−L 47,48 and Ni−R 23,57,58 states have been reported based on spectroscopic signatures, indicating different structural isomers.For instance, temperature-dependent FTIR studies of the soluble [NiFe] hydrogenases observed protonation/deprotonation of the residue homologous to Cys374 L (Cys546 in DvMF) with an ΔH and ΔS of 1.5 ± 0.8 kcal mol −1 and 6.1 ± 10.3 kcal mol −1 K −1 , respectively, suggesting that the cysteine residue can indeed undergo protonation change in the Ni−L state. 47This compares well with our finding on exergonic proton transfer from Glu21 L to Cys68 L or Cys374 L in the Ni−L state.Similarly, a recent IR and EPR investigation on a regulatory [NiFe]-hydrogenase from Cupriavidus necator proposed that the interconversion between the Ni−L 1 and Ni−L 2 forms does not require the breaking of covalent bonds, 48 which is consistent with our finding of two isoenergetic rotamers (3Y2 and 3B) of the protonated Cys374 L in the Ni−L state (Figure 5A).Interestingly, Greene et al. 59 found based on the FTIR and absorption spectroscopic experiments on a soluble hydrogenase from Pyrococcus furiosus, two distinct forms of Ni−L.Our work thus further corroborates these findings on the active participation of different degenerate forms of Ni−L in Ni−SI a → Ni−C transition, and provides a structural assignment for these states.Similar to the Ni−L state, at least three unique spectroscopic signals have been identified for the Ni−R state, implying the coexistence of multiple metastable species.Based on our findings, these states could arise from the different rotamers of the protonated Cys374 L (5E2 LS and 4B LS ), from the different H 2 binding modes, and/or the dihydride-bound state (4B1 LS and 4D LS ) (Figure 5A).Our work thus provides a structural proposal for these spectroscopically characterized states and further highlights the involvement of several spin and conformational states during the catalytic cycle.
As discussed above, our study reveals striking similarities between Mbh and respiratory Complex I, such as conformational changes in the β1-β2 loop, the TM1−2 (MbhI), TM5− 6 (MbhM), and the PSST loop (MbhJ) that are conserved within the superfamily, while the MbhH subunit is homologous to the antiporter-like Nqo12 subunit of Complex I. We suggest that the β1-β2 loop plays a critical role in H 2 catalysis by shuttling protons from the bulk toward the [NiFe] active site, while enabling a redox signal propagation toward the membrane domain that triggers ion pumping across the membrane. 5he long-range signal transduction between the electron transfer activity in the hydrophilic domain and the ion transport in the membrane domain could be achieved through the redox state of the [NiFe] cluster and the protonation state of Cys374 L and Glu21 L .For each catalytic turnover, the conformational changes could be triggered during the Ni−C → Ni−R transition via motion in the network of loops (Figure 5B).The primary coupling event could involve an outward flip of Glu21 L and the subsequent contraction of the β1-β2 loop, which further leads to changes in the ion pairs in TM5−6 (MbhM), PSST (MbhJ), and the TM1−2 loop (Figure 4H).We note the TM1−2 loop shows a lower sequence conservation (ca.20%) relative to the same region in Complex I (Figure S31).In this regard, we note that Asp379 L is conserved in Mbh and Complex I, but not present in hydrogenases, suggesting that the Lys24/Asp379 interaction of MbhL could be important for energy transduction (Figure 4E).In contrast, Glu49 I is unique for Mbh (Figure 4F) and forms an ion pair with Lys40 L , while MbhM (NuoH in Complex I) comprises the unique Glu216 M that interacts with Arg64 J (Figure 4G).The identified electrostatic network provides an important basis for future mutagenesis studies.

■ CONCLUSIONS
The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus, a member of the Complex I superfamily, serves as an intriguing system for studying the interplay between [NiFe]enabled H 2 catalysis and ion transport and its evolutionary relation to Complex I, which catalyzes quinone reduction.By combining density functional theory (DFT) with correlated ab initio methods and atomistic molecular dynamics simulations, we derived key insight into the molecular mechanism of H 2 evolution in Mbh, which has so far been missing, together with a detailed analysis of energetics and dynamics linked to this process.Our findings on the [NiFe] catalysis provide insight into the structures of the Ni−L and Ni−R states, 23,48 the involvement of various spin states during catalysis, as well as a rationale for the activation of the Glu and His proton pathways 56 that is likely to apply also for soluble [NiFe] hydrogenases.
We suggested that Mbh employs redox-driven conformational changes similar to those of Complex I, particularly around the conserved β1-β2 loop that could be mechanistically important for transducing the redox energy into ion transport across the membrane.Moreover, we suggest that Mbh employs electric field effects formed by redox and protonation changes−a mechanism that could serve as a general energy transduction principle in nature.

■ MATERIALS AND METHODS
Quantum Chemical Models.Quantum chemical DFT models were built to probe the geometric and electronic structure of the [NiFe] active site of Mbh.The DFT models were built by combining conserved parts of a high-resolution Journal of the American Chemical Society X-ray structure (at 0.89 Å resolution, PDB ID: 4U9H) of the soluble [NiFe] hydrogenase from DvMF, resolved in the Ni−R state, 36 together with our MD-relaxed system of Mbh, 5 where we substituted all residues that are unique for Mbh (Figure 1B).The central core of the model contained Fe, Ni, the hydride, two CN-and one CO ligands, and all residues in the first and second coordination spheres (Asn36 J , Glu21 L , Cys68 L -Gly69 L -Ile70 L -Cys71 L , His75 L , Ser110 L , Ala318 L -Pro319 L -Arg320 L , Leu323 L , His325 L , Glu343 L -Pro344 L -Thr345 L , Asp372 L , Cys374 L -Leu375 L -Ser376 L -Cys377 L , and a water molecule) (Figure 1B).The residues with a truncated backbone were terminated by a methyl group (−CH 3 ) at the C a atom.The C a atoms and the linker hydrogen atoms of residues were fixed at their X-ray positions during the geometry optimization.The model contains a total of 264 atoms.All geometry optimizations (ground state, transition state, product states) were performed at the TPSSh 60 level with the central core of the active site comprising of Fe, Ni, S, two CN-and CO assigned def2-TZVP basis set, 61 while the rest of the atoms were described with the def2-SVP basis sets.We also applied the multipole accelerated resolution of identity (MARI-J) approximation 62 during the optimization protocol, whereas dispersion effects were included using the empirical dispersion correction with Becke-Johnson damping (D3-BJ). 63 higher DFT integration grid (m4) and tighter SCF thresholds were used throughout (scfconv 8) in all computations.We also employed the implicit solvation COSMO scheme 64,65 with the dielectric constant set to 4.0.The molecular Hessian used for estimation of free energies was computed numerically at the TPSSh/def2-TZVP/def2-SVP level of theory (same as optimization protocol), with scaling factors (0.9615) of the vibrational frequencies based on The reaction energetics reported in this work (see Figures 2,3) are based on the equation shown above, unless stated otherwise.The reaction pathways were optimized between reactant and product states using a method related to the zerotemperature string method, as implemented in TURBOMOLE 7.5.1. 66−70 We used x2c-TZVPP for Fe and Ni, and x2c-TZVP basis set 71 for the rest of the atoms.The choice of B3LYP* functional was chosen based on its performance against the random phase approximation 72 (RPA) method and domain-based pair natural orbital coupled cluster with singles, doubles, and full iterative triples (DLPNO− CCSD(T 1 )) 73−75 calculations to predict spin-state energetics accurately.The final single-point energy and molecular Hessian calculations were performed on a DFT model (Figure S4).The results of the benchmark (geometry, spin state, and reaction energetics) are shown in the Supporting Information.All the calculations were performed with the TURBOMOLE v. 7.5.1. 66he electric field along the proton transfer pathway was computed using optimized structures at the TPSSh/def2-TZVP/def2-SVP level of theory with the TURBOMOLE v. 7.5.1 suite.The electric field contributions of the water molecule between Glu21 L /Cys374 L were omitted from the DFT models.
Molecular Dynamics Simulations.Atomistic molecular dynamics simulations of the Mbh (PDB ID: 6CFW) 1 model were performed using the CHARMM36 force field for protein/ lipids and water.Parameters for the FeS clusters and [NiFe] in different catalytic states (see Tables S22, S23) were derived from in-house DFT calculations (https://github.com/Kaila-Lab/ff_parameters),with the remaining system treated using the CHARMM36m force field. 76The system was embedded in a 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphoinositol (PYPI) membrane using CHARMM-GUI 77 modeling unresolved side chains, 5 as well as different protonation states with lipids in the cleft between MbhM and MbhH.The model was embedded in a 200 × 100 × 168 Å 3 box comprising TIP3P water molecules and ions to mimic a 250 mM NaCl concentration.The [NiFe] catalytic site was modeled into Mbh using the high-resolution soluble hydrogenase (PDB ID: 4U9H). 36Initial protonation states were obtained from Poisson−Boltzmann electrostatic calculations with Monte Carlo sampling, 5 based on models with the cofactors treated in their oxidized state (see SI Appendix, Table S24, for a list of residues with nonstandard protonation state).All MD simulations were performed in an NPT ensemble at T = 310 K and p = 1 atm, using a 2 fs integration time step, and with electrostatics modeled using the particle mesh Ewald (PME) method.The system was gradually relaxed for 10 ns with harmonic restraints of 2 kcal mol −1 Å −2 on all protein and cofactor heavy atoms, followed by 10 ns with harmonic restraints of 2 kcal mol −1 Å −2 on all protein backbone heavy atoms, and by 10 ns equilibration with weak (0.5 kcal mol −1 Å −2 ) restraints on all C α atoms and 0.5 μs production runs.All classical MD simulations (3.5 μs in total) were performed using NAMD2 (v.2.12/2.13)for equilibration and NAMD3 78 for production runs.The simulations were analyzed using VMD. 79Multiple sequence alignment (MSA) for soluble hydrogenases and the Complex I superfamily was done with ClustalW 80 and visualized with Jalview. 81
Extended methods, geometric and molecular energetics benchmark, MD simulation data, DFT models, reaction profiles, geometric analysis, additional information of

Figure 1 .
Figure 1.Structure, function, and catalytic cycle of Mbh.(A) Overall structure and function of Mbh, showing the hydrophilic domain, responsible for ferredoxin (Fd)-driven H + reduction to H 2 gas, and the membrane domain, responsible for proton pumping and Na + /H + exchange across the membrane (PDB ID: 6CFW 1 ).Arrows along the transmembrane region show subunits that could be responsible for the ion transport, whereas the stoichiometry and directionality are currently unknown. 5,8,10Redox active cofactors responsible for the electron transfer from Fd to the [NiFe] center are also shown.(B) DFT model used in the present work to study the mechanism of [NiFe] catalysis, and (C) catalytic states associated with the H 2 catalysis in [NiFe] hydrogenases. 15(D) Structure of the [NiFe] active site, the β1-β2 loop, harboring Glu21 L , and a catalytically active water molecule, hydrogen bonding with Glu21 L and Cys374 L of subunit MbhL.

Figure 2 .
Figure 2. Reaction energetics in the Ni−SI a , Ni−L, and Ni−C states.(A) Energetics of proton transfer from Glu21 L to Cys374 L in the low spin (LS) and high spin (HS) forms of the Ni−SI a state.The transferred proton is highlighted in green.(B) Top: spin density distribution in the 1B HS state, and bottom: overlay of optimized LS and HS models of the Ni−SI a state.(C) Dependence of the reaction energy and barrier on an electric field applied along the proton transfer coordinate.(D) Energy profile for proton transfer from Glu21 L to Cys374 L in the Ni−L state and subsequent transition to the Ni−C state.The transferred proton is highlighted in green.(E) Spin density distribution in the Ni−L (3B) and Ni−C (3D) states, with marked Ni−Fe distances.(F) Electric field strength along the H 2 O−Cys374 L proton transfer coordinate in the Ni−SI a state (1B LS ) and in the one-electron-reduced Ni−L state.Inset: the electric field vectors along the proton transfer coordinate for the Ni−SI a (Ni II ) and Ni−L states (Ni I ).(G) Electric field difference in the Ni−SI a minus Ni−L states.Inset: the electric field vectors point toward the sulfur atom of the Cys374 L upon reduction of Ni II .

Figure 3 .
Figure 3. Free energy profile of H 2 formation in the Ni−R state.(A) Free energy profile of the H 2 formation in the low spin (LS, in black) and high spin (HS, in red) configurations of the Ni−R state.Energetics of proton transfer via the histidine (His75 L ) and glutamate (Glu21 L ) pathways.(B) Structure of different intermediates involved in the H 2 formation step.His75 L can remain protonated (His + ), while the Glu pathway is used for the second proton delivery (see Figures S23−S25).The structures of the transition states are shown in Figuress S14−S15.

Figure 4 .
Figure 4. Redox-triggered conformational coupling between redox catalysis and loop dynamics.(A) Clustering of water molecules between the Glu21 L and the [NiFe] cluster, and the distance between Cys374 L and Glu21 L in different catalytic states from MD simulations.(B) Population of "flipped-in" conformation of Glu21 L in different catalytic states.Ni−SI a ' and Ni−SI a differ in the modeled redox states of the Fd and Fe−S clusters (see Table S23), while in the Ni−C* state, Glu20 L is protonated and Glu21 L deprotonated (and vice versa in the Ni−C state).(C) Water occupancy between Glu21 L and Cys374 L in MD simulations.(D) Structure and location of the loop network and the [NiFe] cluster of Mbh.(E) Effect of the Glu21 L "flip" on the ion-pair dynamics in different catalytic states.The outward motion of Glu21 L in the Ni−C state favors dissociation of the ion pair with Lys24 L and Asp379 L , and contraction of the β1-β2 loop (see panel H).(F) Interaction of the TM1−2 loop (MbhI) and the β1-β2 loop (MbhL).The contraction of β1-β2 leads to dissociation of an ion-paired network between different subunits and an outward motion of the TM1−2 loop (see also panel H).(G) Interactions among the TM1−2 loop, the TM5−6 loop, and the PSST loop.The TM1−2 loop forms an ion pair among Lys43 I , Glu216 M , Glu221 M , and Arg64 J .(H) Conformational changes in loops upon conformational changes in Glu21 L ("inward"−dark colors; and "outward"−light colors orientations).Subsequent conformational changes in the TM1−2 loop are also shown ("open" vs "close").Details of the redox and protonation states in various states are shown in TablesS22 and S23.

Figure 5 .
Figure 5. Structural models of intermediate states in the catalytic cycle and proposed coupling elements.(A) Structural models for the Ni−SI a , Ni− L, Ni−C, and Ni−R states in Mbh.The catalysis proceeds via the one-electron reduction of Ni−SI a and the subsequent generation of a directed electric field gradient that drives the proton transfer from Glu21 L to Cys374 L /Cys68 L , forming the Ni−L state.The high spin (HS) state is more stable relative to the low spin (LS) form for the Ni−SI a state (G HS < G LS ).Different structures exist for Ni−L, with energetically degenerate rotamers of the protonated Cys374 L or protonated Cys68 L .Subsequent oxidation of Ni I leads to the Ni−C state (Ni III ) with a bridging hydride (μ-H − ).Subsequent one-electron reduction of the Ni−C state forms the Ni−R state (Ni II ), followed by proton transfer along the Glu or His pathways that result in the protonation of Cys374 L or Cys377 L , respectively.Possible representative structures of the Ni−R state are shown along the H 2 formation reaction (see also Figure 3).H 2 formation in the Ni−R state is favored by the LS Ni II form (G LS ≪ G HS ).(B) Suggested redox-driven long-range coupling elements in Mbh.Top inset: Primary signal transduction steps induced by the [NiFe] redox chemistry involve: [1] one-electron reduction of the [NiFe] by the proximal FeS cluster, which induces a directed electric field toward Cys374 L that[2] leads to proton transfer from Glu21 L to Cys374 L .The "flipped-in" orientation of Glu21 L favors the proton transfer and formation of an ion-paired interaction between Lys24 L and Asp379 L .Bottom inset: Following the proton transfer step:[1] Glu21 L flips into its "outward" conformation, which[2] favors dissociation of the Lys24 L -Asp379 L ion pair.Subsequently, the β1-β2 loop undergoes a contraction[3], which in turn favors the dissociation of ion pairs between the TM1−2 and the β1-β2 loop[4].This cascade leads to conformational changes in the TM1−2 loop, which moves toward the membrane domain.The motion is supported by contacts between Lys53 I and Glu375 H .[6]These conformational changes also lead to the formation of ion pairs between TM1−2 (MbhI), PSST (MbhJ), and TM5−6 (MbhM) loops.The interaction of the TM1−2 loop with the MbhH subunit via Lys53 I -Glu375 H is shown, together with key residues along the putative proton pathway in MbhH.