Reactivation of the Ready and Unready Oxidized States of [NiFe

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Reactivation of the Ready and Unready Oxidized States of [NiFe]Hydrogenases: Mechanistic Insights from DFT Calculations Raffaella Breglia,† Claudio Greco,† Piercarlo Fantucci,‡ Luca De Gioia,‡ and Maurizio Bruschi*,† †

Department of Earth and Environmental Science and ‡Department of Biotechnology and Biosciences, University of Milano Bicocca, 20126 Milan, Italy

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S Supporting Information *

ABSTRACT: The apparently simple dihydrogen formation from protons and electrons (2H+ + 2e− ⇄ H2) is one of the most challenging reactions in nature. It is catalyzed by metalloenzymes of amazing complexity, called hydrogenases. A better understanding of the chemistry of these enzymes, especially that of the [NiFe]-hydrogenases subgroup, has important implications for production of H2 as alternative sustainable fuel. In this work, reactivation mechanism of the oxidized and inactive Ni−B and Ni−A states of the [NiFe]hydrogenases active site has been investigated using density functional theory. Results obtained from this study show that one-electron reduction and protonation of the active site promote the removal of the bridging hydroxide ligand contained in Ni−B and Ni−A. However, this process is sufficient to activate only the Ni−B state. H2 binding to the active site is required to convert Ni−A to the active Ni−SIa state. Here, we also propose a reasonable structure for the spectroscopically well-characterized Ni−SIr and Ni−SU species, formed respectively from the one-electron reduction of Ni−B and Ni−A. Ni−SIr, depending on the pH at which the reaction occurs, features a bridging hydroxide ligand or a water molecule terminally coordinated to the Ni atom, whereas in Ni−SU a water molecule is terminally coordinated to the Fe atom, and the Cys64 residue is oxidized to sulfenate. The sulfenate oxygen atom in the Ni−A state affects the stereoelectronic properties of the binuclear cluster by modifying the coordination geometry of Ni, and consequently, by switching the regiochemistry of H2O and H2 binding from the Ni to the Fe atom. This effect is predicted to be at the origin of the different reactivation kinetics of the oxidized and inactive Ni−B and Ni−A states.

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INTRODUCTION Concerns about the depletion of fossil fuels and the growing awareness of the impact of greenhouse gas emissions on global climate change have increased interest of the scientific community in the search for renewable and sustainable energy sources.1,2 In this regard, molecular hydrogen has attracted particular attention as alternative fuel because of its environmental benignity and high energy efficiency.3 However, several challenges and obstacles currently limit the viability of dihydrogen as a serious replacement for fossil fuels. These include, but are not limited to, sustainable production issues. Currently, hydrogen is in fact mainly produced from natural gas and petroleum fractions in highly energy-consuming and not environmentally friendly processes. Carbon-neutral H2 production through electrolysis of water, on the other hand, is limited by high cost and availability of platinum group metals which are required as catalysts.4 New economically competitive ways for sustainable H2 production need therefore to be developed. In this context, one of the most promising solutions is the photobiological production of H2 by photoautotrophic organisms, such as green microalgae and cyanobacteria, © XXXX American Chemical Society

which have evolved formidable catalysts for the production of H2 and O2 from the most abundant of the natural resources, sunlight and water, with low- to net-zero carbon emissions (H2O → H2 + 1/2O2). This process, also referred to as biophotolysis, involves two steps: the splitting of water into molecular oxygen, protons, and electrons by photosynthesis (H 2 O → 2H+ + 2e− + 1 / 2 O 2 ) and the subsequent recombination of the released protons and electrons into molecular hydrogen (2H+ + 2e− → H2). However, widespread adoption of this technology is so far impeded by the O2sensitivity of hydrogenases, the natural catalysts of the H+/H2 interconversion.5−9 Oxygen, in addition to being very abundant in our atmosphere, is generated as a byproduct during the water-splitting process. Therefore, maintaining hydrogenase activity in the presence of O2 or designing efficient O2-tolerant biomimetic catalysts is crucial for biotechnological H2 production.10,11,20−24,12−19 In this context, increasing efforts have been devoted to the better understanding of the hydrogenase chemistry. Received: August 20, 2018

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DOI: 10.1021/acs.inorgchem.8b02348 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Figure 1. Cartoon representation of the X-ray crystal structure of the [NiFe]-hydrogenase from Allochromatium vinosum (PDB: 3MYR). The small subunit (green) harbors two [Fe4S4] clusters and one [Fe3S4] cluster which transfer electrons to and from the active site, which is contained in the large subunit (orange). The latter and the residues included in the largest DFT model used in this work are shown enlarged on the right (ball and stick representation). Atoms are colored according to the following scheme: nickel, green; iron, orange; nitrogen, blue; oxygen, red; sulfur, yellow; carbon, tan; hydrogen, white. For the sake of clarity, aliphatic hydrogen atoms are not shown.

Scheme 1. Overview of the Different Redox States of the NiFe Active Site of the [NiFe]-Hydrogenase Enzymea

a

Active and inactive forms are respectively indicated by green and red labels. States marked with an asterisk show an S = 1/2 EPR signal.

genases the most promising candidates for large-scale photobiological hydrogen production. Standard [NiFe]-hydrogenases are heterodimeric proteins composed by a large subunit harboring the active site and a small subunit containing an electron-transfer relay of two [Fe4S4] clusters and one [Fe3S4] cluster (see Figure 1). The active site consists of a binuclear cluster, in which one Ni and one Fe atom are bridged by two cysteine residues (Cys64 and Cys558 in the residue numbering of the enzyme from Allochromatium vinosum). Two further cysteines are terminally bound to the Ni atom (Cys61 and Cys555), while one carbonyl and two cyanide ligands are coordinated to the Fe atom. A third bridging ligand, whose nature depends on the enzyme redox state, can also be present. In O2-resistant [NiFe]-hydrogenases, all [FeS] clusters are of the [Fe4S4] type and one of the two terminal cysteine residues of the active site is substituted by a selenocysteine.36 The active site of the O2tolerant enzymes is instead the same as that of O2-sensitive

Hydrogenases, according to the active site composition, are subclassified into [Fe]-, [FeFe]-, and [NiFe]-hydrogenases.7,25−27 [Fe]-hydrogenases, however, catalyze the heterolytic cleavage of H2 (H2 → H+ + H−) and not its reversible conversion to protons and electrons.28−31 Despite their reversibility, [FeFe]-hydrogenases usually work in the direction of the H2 evolution, whereas [NiFe]-hydrogenases are usually involved in H2 oxidation.32 Another significant difference among the three classes of hydrogenases is their reactivity toward O2. [Fe]- and [FeFe]-hydrogenases are irreversibly inactivated by even traces of O2, while [NiFe]-hydrogenases are more resistant to oxidation and can be further classified as O2-tolerant, O2-resistant, and O2-sensitive. The latter group (referred to as the standard enzymes), upon exposure to O2, leads to a mixture of two different oxidized inactive forms, the so-called Ni−B and Ni−A states, that can be reactivated upon reduction.33−35 The latter property makes [NiFe]-hydroB


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Inorganic Chemistry [NiFe]-hydrogenases, but they differ from the conventional [NiFe] enzyme for the replacement of a [Fe4S4] cluster by a modified [Fe4S3] cluster.37−40 Several active and inactive forms of the active site of [NiFe]hydrogenases have been experimentally characterized by infrared (IR) and electron paramagnetic resonance (EPR) spectroscopy (see Scheme 1).41−45 For what concerns the active forms, three redox states involved in the catalytic cycle have been identified: the EPR-silent states Ni−R and Ni−SIa, in which both the Ni and Fe atoms attain the +2 oxidation state, and the EPR-active Ni−C form in which the Ni atom is formally oxidized to NiIII. Ni−SIa is characterized by one vacant coordination position between the metal atoms, where H2 was proposed to bind, whereas Ni−R and Ni−C feature a bridging hydride ligand. Upon illumination at cryogenic temperatures of Ni−C, a new paramagnetic state named as Ni−L is generated. Ni−L is generally considered to be a NiIFeII species with a vacant bridging position resulting from the dissociation of the hydride ligand as a proton, which presumably moves to one of the sulfur atoms coordinated to Ni, and two electrons that reduce the NiIII ion to NiI. Conversely, in both inactive and paramagnetic Ni−B and Ni− A states the active site attains the NiIIIFeII redox state and a hydroxide ligand bridges the metal ions.46,47 The structural difference between these two states, responsible for the different IR and EPR signatures,48 has remained elusive until recently when crystallographic and DFT studies have confidently assigned to Ni−A a structure featuring a bridging cysteine residue oxidized to bridging sulfenate, in addition to the hydroxide ligand bridging to the metal atoms.49,50 Oxidized Ni−B and Ni−A states require reductive activation that involves a structural reorganization of the active site from the oxidized form to the species that is catalytically active for H2uptake. The rate of this activation process is however markedly slower for Ni−A than for Ni−B.51 The Ni−B state upon addition of H2 or low-potential electron donors under anaerobic conditions takes few minutes to restore the enzyme activity; therefore, it is also called the “ready” state. The oneelectron reduction of the Ni−B form yields an EPR-silent species referred to as Ni−SIr (silent ready), which is promptly converted to the Ni−SIa (silent active) form; the latter is an intermediate of the catalytic cycle. The distinction between these two silent states (ready and active) has been attributed to protonation and subsequent dissociation of the oxygenic bridging ligand from the active site.52 Conversely, the Ni−A state is also known as the “unready” state because it is reduced within minutes to the EPR-silent Ni−SU (silent unready) state, but several hours under H2 are required to return to the active Ni−SIa state. Despite the spectroscopic and kinetic evidence, many aspects of the reactivation mechanism of oxidized [NiFe]hydrogenases, such as the order of electron and proton transfers, the role of H2, and the rate-determining step, are still poorly understood. In this context, theoretical methods, particularly density functional theory (DFT), may be extremely helpful to shed more light on these aspects. DFT calculations have been used to propose possible reaction mechanisms in which Ni−B and Ni−A are converted into the catalytically active Ni−SIa state.53−55 However, results from such calculations cannot be considered exhaustive because only the bimetallic cluster of the [NiFe]-hydrogenase active site has been included in the DFT models. Moreover, the reactivation mechanism of the Ni−A state was proposed by Jayapal et al.54

in 2006 when the more recent and better resolved X-ray structure assigned to Ni−A, featuring a bridging hydroxide and the Cys64 residue oxidized to bridging sulfenate, was not yet available.49 In this study, Ni−A was incorrectly modeled with a hydroperoxide ligand bridging the two metal ions.56 For these reasons, we decided to perform a detailed DFT study on a very large model of the active site of standard [NiFe]-hydrogenase that has already proved to allow for a correct description of the Ni−A state.50 Results from this study lead us to propose a novel Ni−B and Ni−A reactivation mechanism which accounts for their different activation kinetics and relates them to their structural features.

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METHODS

Active Site Model. The active site model used in this work was constructed on the basis of the crystal structure of the O2-sensitive [NiFe]-hydrogenase from Allochromatium vinosum (Protein Databank (PDB) code: 3MYR).57 As shown in Figure 1 (right), it contains 288 atoms and consists of the heterobimetallic cluster and selected residues of its first- and second-coordination-spheres that in the following are numbered according to the 3MYR structure. Specifically, the model includes the NiFe site and all atoms of its direct ligands, namely, one CO, two CN−, and four cysteine residues. Two of these cysteines (Cys64 and Cys558) bridge the two metal ions, whereas the other two (Cys61 and Cys555) are terminally coordinated to the Ni atom. These residues belong to two different Ni-binding motifs (Cys61−Gly62−Val63−Cys64 and Cys555−Ile556−Ala557− Cys558). Each motif contains one terminal and one bridging cysteine separated by two other amino acids that are also contained in the model. However, only the backbone atoms of Ile556 and Ala557 are included in the model because their side chains are far from the active site. In contrast, the Val63 side chain is included in the model since it is oriented toward the active site. Selected backbone atoms of Ile60, Thr65, Pro554, and Ala559 are also added to the model to terminate the two motif chains. The model contains Val67, Leu490, Val507, Val508, and Pro509 because these residues form a hydrophobic pocket around the Fe(CO)(CN)2 group. In addition, Ile13, Glu14, Gly15, Thr17, His68, Asp103, Arg487, Ser 510, and Asp553 are also included in the model since their side chains establish hydrogen bonds with the amino acids belonging to the two Ni-binding motifs or the diatomic ligands coordinated to the Fe atom. His68 is also added to the model due to its proximity to the bimetallic cluster, and is always modeled as the neutral Nε−H tautomer because among all possible protonation states for this residue the latter is demonstrated to be the form which better overlaps with the experimental structure of the Desulfovibrio vulgaris Miyazaki F [NiFe]-hydrogenase.58 In contrast, Glu14 is modeled both as glutamate and glutamic acid since its carboxyl group is very close to the sulfur atom of Cys555; therefore, it can play a crucial role in proton transfer events from/to the NiFe cluster.59−61 As a final step, atoms at the boundary of the model for which bonds have been truncated are saturated with hydrogens. The resulting model has been previously validated in our laboratories; thanks to the presence of numerous residues belonging to the second coordination sphere, it was proven to correctly describe the conformation of the Ni atom at the active site.62 Furthermore, it allowed us to accurately evaluate the relative stabilities of the inactive oxidized forms50 and to discuss with reasonable confidence mechanistic routes.63 A medium model, containing up to 138 atoms, is also considered. It contains the NiFe cluster and its first coordination sphere. In the second coordination sphere, the model includes selected atoms of Glu14, Gly62, Val63, Thr17, His68, Asp103, Arg487, Pro509, Ser510, Asp553, Ile556, and Ala557. Initial coordinates are taken from the corresponding optimized structure of the large-sized model. The Supporting Information contains a detailed list of the atoms composing the two models and of the atoms that during geometry optimizations are fixed at their respective X-ray positions in order to C


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Inorganic Chemistry Scheme 2. Schematic Representation of the Ni−B Activation Pathway Leading to the Ni−SIa Statea

a

The most plausible mechanism is indicated by bold arrows. For those species in which Glu14 is not explicitly drawn, it is modeled as glutamate. Species depicted in grey are not calculated as stable intermediates in this work. Energy differences are in kcal/mol. Values in italic are calculated using the medium-sized model. Superscripts a and b denote dissociation energies of a H2O molecule from the metallic site to a position in which it is hydrogen bonded to Arg487 or to Cys61, Cys64, and His68, respectively. avoid unrealistic distortions at the boundary of the model (see Table S1). Computational Details. All calculations are performed in the DFT framework with the TURBOMOLE program package64 using the BP8665,66 functional in conjunction with the resolution-of-theidentity (RI) technique.67 In both models, an all-electron valence triple-ζ basis set with polarization functions (TZVP)68 is used for Ni, Fe, the atoms of the first coordination sphere, and the Glu14 residue. For all other atoms, the smaller SVP and SV(P) double-ζ basis sets69 are used instead in the large and the medium model, respectively (for details, see Table S1). Geometry optimizations are carried out using the conductor-like screening model (COSMO)70,71 with a polarizable continuum medium at ε = 472−74 to account for the effects of the surrounding protein matrix on the stereoelectronic properties of the active site. Therefore, the values discussed in the text are electronic energy differences without thermal corrections and including solvation energies computed with the COSMO scheme implemented in TURBOMOLE. It is worth noting that energy differences calculated in vacuum and in the polarizable continuum medium are very similar indicating that the size of the models is large enough to be insensitive to nonspecific polarizing effects of the protein environment. Since the results obtained by previous investigations with and without dispersion interactions correction given by the DFT-D3 Grimme scheme75−77 as implemented in TURBOMOLE64 are consistent with each other, dispersion interactions are not included in the calculations. For species containing the Ni atom in the NiII redox state both singlet (S = 0) and triplet (S = 1) spin states are calculated, while for the NiIII ion only the low spin state (S = 1/2) is considered. In several cases, to investigate the appropriate spin coupling of the system, the broken symmetry (BS) approach is also used; the latter was introduced by Noodleman et al.78 and consists of the localization of opposite spins of the monodeterminant wave function in different parts of the molecule. Geometry optimizations of all minimum energy structures are performed using the largest model, whereas optimizations of the transition states are performed using the medium model. Transition states search is performed according to a pseudo-Newton−Raphson procedure and applying the trust-region image minimization method,79 which is designed to maximize the energy along one of the eigenvectors (i.e., the chemically relevant one) and minimize it in all other directions. The presence of constrained atoms resulted in the occurrence of several imaginary frequencies corresponding to torsional vibrational modes involving the constrained atoms. However, the eigenvector corresponding to the reaction coordinate

was easily identified as the one with an eigenvalue much lower than the other negative eigenvalues. Entire reaction profiles for dissociation of the bridging hydroxide ligand as water molecule from Ni−B and Ni−A are obtained by generating several structures connecting reactants and products. In this respect, the distance between the oxygen atom of H2O and the either the Ni or the Fe atom in the active site is assumed as linear reaction coordinate. Geometry optimizations of such models are carried out fixing this distance to certain values and optimizing all other parameters to their most favorable values. The higher energy structure obtained along the reaction coordinate is assumed to be the transition state for the corresponding reaction. Nomenclature. In the following, computational models of the investigated species are labeled according to the n-OxHy(k,j) general scheme where n is a generic number referring to the binding mode of the various ligands coordinated to the active site. The x and y subscripts specify the number of the oxygen and hydrogen atoms contained in such ligands, whereas the k and j superscripts indicate the charge and the spin state of the considered species, respectively. The OxHy tag is omitted if no ligands are bound to the enzyme active site. Conversely, the GluH tag follows the n-OxHy(k,j) label if the Glu14 residue attains neutral state. Energies of all species investigated in this work are shown in Table S2.

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RESULTS X-ray diffraction studies showed that the main structural difference at the level of the active site between the oxidized inactive and the reduced active enzymes is that in the latter the oxygenic species that bridges the metals is absent and that the Ni−Fe distance is ca. 0.25 Å shorter.36 On the other hand, EPR experiments and theoretical studies suggested that for both the inactive Ni−B and Ni−A states the one-electron reduction of NiIII to NiII is the first step of the reactivation process.48,80,81 The monoreduced states of Ni−B and Ni−A, namely, Ni−SIr and Ni−SU, respectively, are both EPR-silent and catalytically inactive, but they can be distinguished on the basis of the IR bands of their CO and CN ligands.52 The combination of these results supports the hypothesis that the one-electron reduction of the oxidized states promotes the removal of the bridging oxygenic species, allowing H2 binding to the active site and catalytic turnover.36 Guided by these data, the one-electron reduction of the active site has been investigated as the first step of the activation process of Ni−B and Ni−A. However, since both states were reported to be D


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Inorganic Chemistry

Figure 2. Schematic representation of the geometries of selected forms of the [NiFe]-hydrogenase active site involved in the Ni−B reactivation mechanism (atoms are color-coded as in Figure 1). Selected interatomic distances are given in Å. For the sake of clarity, aliphatic hydrogen atoms are not shown.

Figure 3. Energy profile for (a) 2-OH2(−4,1) → 2+OH2-A(−4,1) and (b) 2-O2H2(−4,1) → 2+O2H2-A(−4,1) reactions, obtained generating several structures connecting the reactant and the product using the medium model according to the procedure described in the “Methods” section. Schematic representation of the geometries of minimum and maximum energy structures along the reaction coordinate (Ni−OOH2 and Fe−OOH2 distances, respectively) are shown in the circular frames. Selected interatomic distances are given in Å. Energies are in kcal/mol.

activated also upon incubation with H2, the direct binding of H2 to the active site has been investigated as well. Activation of Ni−B. Infrared spectroscopy studies suggest the existence of two forms of the Ni−SIr state (the oneelectron reduced state of Ni−B) that differ for their protonation state at the active site.52,82 Reduction of Ni−B at 2 °C shifts the ν(CO) band from 1943 to 1931 cm−1 at pH 6 (Ni−SIr‑1931) or 1910 cm−1 at pH 9 (Ni−SIr‑1910). At low pH, EXAFS studies indicate that the removal of the bridging oxygen species and the shortening of the Ni−Fe distance from 2.85 to 2.60 Å has already occurred.83 Albracht et al.84,85 suggested that the bridging OH− is still present in the Ni−SIr state prepared at 2 °C and pH 9, but it becomes protonated at low pH leading to the formation of a water molecule. It was assumed that at 2 °C this H2O molecule remains captured in the active-site pocket and sterically hinders the reaction with H2 whereas at room temperature it is released allowing the enzyme activation. On the basis of these experimental observations, the reactivation mechanism of the Ni−B state is proposed to involve reduction of the active site, protonation, and dissociation of the oxygenic bridging ligand. Results obtained from our calculations corroborate this hypothesis and establish the order of events. In particular, our results lead us to suggest

that the reactivation pathway of the Ni−B state occurs following the mechanism shown in Scheme 2, according to which monoelectronic reduction and protonation of Ni−B, followed by the dissociation of the formed water molecule, yields the active Ni−SIa state. Reduction of Ni−B, 1-OH(−4,2) in Scheme 2, leads to the diamagnetic (i.e., singlet) state 1-OH(−5,1), in which the NiIII atom is reduced to NiII and the hydroxide ligand still bridges the Ni and Fe atoms (see Figure 2). It is worth noting that the corresponding high-spin triplet state, 1-OH(−5,3), is less stable by 3.9 kcal/mol. Subsequent protonation of the active site produces a water molecule that moves from the bridging position to the terminal coordination site of the Ni atom. In this new species (2-OH2(−4,1) in Scheme 2 and Figure 2) the Ni atom features a distorted square-based pyramidal geometry; the OH2O−Ni and OH2O−Fe distances are 2.10 and 3.84 Å, respectively, whereas the Ni−Fe bond length is 2.63 Å. The isomer in which H2O bridges the two metal ions is 16.7 kcal/ mol higher in energy than the terminal one, the latter species being largely stabilized by the formation of a strong H-bond between H2O and the guanidinium group of Arg487. 2OH2(−4,1) is also more stable by 11.8 kcal/mol than the corresponding triplet state, 2-OH2(−4,3). The feasibility of formation of a H2O molecule is supported by the fact that the E


Article

Inorganic Chemistry Scheme 3. Schematic Representation of the Ni−A Activation Pathway Leading to the Ni−SIa Statea

a

The most plausible mechanism is indicated by bold arrows. (a) One-electron reduction and protonation of the Ni−A active state, and the consequent release of the hydroxide ligand are marked by a yellow background, while subsequent H2 binding and cleavage (b) is depicted with a red background. Conversely, the green background denotes the release of the oxygen atom of the sulfenate group (c). For those species in which Glu14 is not explicitly drawn, it is modeled as glutamate. Superscripts a and b denote dissociation energies from the metallic site of a H2O molecule hydrogen bonded to Arg487 (corresponding to 2+OH2-A(−4,1)) or to Cys61, Cys64, and His68 (corresponding to 2+OH2-C(−4,1)), respectively. Species depicted in gray are not calculated as stable intermediates. Values in parentheses are the reaction energy differences calculated for the corresponding steps of the catalytic cycle using the large model, whereas values in italic are calculated using the medium-size model. Energy differences are in kcal/mol.

stable than corresponding reactant 1-OHGluH(−3,2) by 11.5 kcal/mol. Furthermore, 1-OH2(−3,2) is not a stable intermediate since the proton initially placed on the S atom of Cys555 spontaneously migrates to Glu14 during geometry optimization. Finally, once 2-OH2(−4,1) is formed, the water molecule dissociates from the active site with formation of the Ni−SIa state (1(−4,1) in Scheme 2). Several positions near the active site have been considered for the H2O molecule as product of the dissociation process (see Figure S2). In the first position along the dissociation coordinate corresponding to an energy minimum (2+OH2-A (−4,1), see Figure S3), the H2O molecule is about at 3.6 Å from the Ni atom forming a H-bond with the guanidinium group of Arg487. The energy difference between

proton transfer from Glu14, a residue previously proposed to be involved in proton transfer from the bimetallic cluster of the active site,59−61 to the nearby sulfur atom of Cys555 is nearly isoenergetic (1-OHGluH(−4,1) → 1-OH2(−4,1), + 1.7 kcal/mol), whereas the subsequent proton transfer from Cys555 to the bridging OH− ligand is a strongly exoenergetic process (1OH2(−4,1) → 2-OH2(−4,1), −11.3 and −5.0 kcal/mol using the large and the medium models, respectively). In addition, the activation energy calculated for the last proton transfer step is equal to 8.5 kcal/mol (see Figure S1). Notably, analogous calculations carried out on the one-electron-oxidized counterparts of the above species provide results supporting the picture that proton transfer follows the monoelectronic reduction step. Indeed, the H2O-adduct 2-OH2(−3,2) is less F


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Inorganic Chemistry

Figure 4. Schematic representation of the geometries of selected forms of the [NiFe]-hydrogenase active site involved in the Ni−A reactivation mechanism (atoms color-coded as in Figure 1). Selected interatomic distances are given in Å. For the sake of clarity, aliphatic hydrogen atoms are not shown.

2+OH2-A, with H2O in such position, and 2-OH2(−4,1) is equal to about 4 and −2 kcal/mol when calculated using the large and medium models, respectively. Notably, the activation energy for the dissociation of H2O from 2-OH2(−4,1) to give 2+OH2-A(−4,1), evaluated by considering the energy profile as reported in the “Methods” section (see Figure 3a), is as low as 2.6 kcal/mol, with the transition state located for a Ni−OH2 distance of about 2.8 Å. The H2O molecule can then migrate from the position in 2+OH2-A(−4,1) to other adjacent positions. In particular, we found energy minimum configurations for the H2O molecule H-bonded to the carboxylic oxygen atom of Asp103 and the carbonyl oxygen of Val63 (2+OH2-B(−4,1)) and in which H2O results H-bonded to Cys61, Cys64, and His68 (2+OH2-C(−4,1)). These two intermediates are more stable than 2+OH 2 -A (−4,1) by 9.9 and 6.5 kcal/mol, respectively. Notably, the presence of a crystallization water molecule very close to Cys61, Cys64, and His68 in the X-ray 3MYR structure57 supports the hypothesis that these three residues are relevant for the modulation of the interaction between water and the enzyme active site. In fact, it is tempting to suggest that an equilibrium can be established between the species in which a water molecule is coordinated to Ni and that in which H2O is H-bonded to residues near the active site (2OH 2(−4,1) → 2+OH2-C(−4,1), −2.1 kcal/mol). This is compatible with the experimentally observed easy interconversion between Ni−SIr and Ni−SIa at 25 °C.85 According to the mechanism here proposed for the Ni−B activation, the Ni−SIr‑1910 and Ni−SIr‑1931 states, experimentally observed by Bleijlevens et al.52 should correspond to 1OH(−5,1) and 2-OH2(−4,1). They were reported to differ for the protonation state, as stated on the basis of infrared spectroelectrochemical studies. In such context, our calculations interestingly show that reduction and protonation of the Ni−B state to form 2-OH2(−4,1) lead to the removal of the bridging ligand (Fe−O distance = 3.84 Å, Ni−O distance = 2.10 Å) and

to the shortening of the Ni−Fe distance from 2.88 to 2.63 Å, in full agreement with EXAFS data (see Figure 2).83 Since reductive activation processes can take place upon H2 exposure, the reaction of H2 with the active site of the Ni−B state is also investigated. In this respect, Kurkin et al.84 proposed a reactivation pathway in which the Ni−B state, in the presence of H2, is directly converted to the Ni−SIr‑1931 state. According to this mechanism, the bridging hydroxyl group of Ni−B heterolytically cleaves the H2 molecule leading to a water molecule and a hydride ion that is then oxidized resulting in the one-electron reduction of the Ni atom and of one of the auxiliary Fe−S clusters. This hypothesis is apparently supported by the DFT study performed by Jayapal et al.54 according to whom H2 weakly binds the Ni atom in the Ni−B state, with a binding energy of +0.6 kcal/mol. However, besides being slightly higher in energy than the reactants, the obtained species is characterized by a too long distance between the H2 molecule and the active site to be considered a H2 adduct. In agreement with this assumption, our calculations show that the adduct in which H2 binds to the NiIII atom of Ni−B is unstable, resulting in H2 release during geometry optimization, whereas the H2 binding to the Fe atom is energetically disfavored by as much as 15.4 kcal/mol. It should be noted that according to our results the chemical process represented by the Ni−B + H2 → Ni−C + H2O reaction corresponding to the H2 binding and cleavage at the Ni−B active site with subsequent H2O release and Ni−C formation is exoenergetic by about 5 kcal/mol. However, this pathway should be ruled out in consideration of the fact that it does not imply the formation of a diamagnetic intermediate, whereas in experiments the occurrence of the EPR-silent Ni−SIr state is observed instead. Finally, H2 binding to 2-OH2(−4,1) has been investigated since Stein et al.53,55 suggested that H2 cleavage occurs after protonation and monoelectron reduction of the Ni−B state. According to their results, the water molecule coordinating the NiIIFeII site would act as a base for the G


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Inorganic Chemistry

In 2-O2H2(−4,1), the H2O molecule is terminally coordinated to the Fe atom, with Fe−OH2O and the Ni−OH2O distances equal to 2.10 and 2.94 Å, respectively (see Figure 4). H2O binding is stabilized by a weak interaction between one hydrogen atom of water and Ni (Ni- - -H(OH) = 2.06 Å). Indeed, a species with a different orientation of H2O, such that the same hydrogen is at a distance of 2.81 Å from the Ni atom is about 6.5 kcal/mol less stable than 2-O 2 H 2 (−4,1) . Interestingly, in 2-O2H2(−4,1) the tetracoordinated Ni atom features a geometry in which the Sa−Ni−Sb and Sc−Ni−OSd angles are equal to 143 and 161°, respectively (see Scheme 4).

heterolytic cleavage of H2 during dihydrogen oxidation catalysis. However, our calculations show that H2 binding to the active site is energetically disfavored also in this case. On the basis of these considerations, it is possible to confidently conclude that the reactivation pathway of Ni−B does not involve the direct interaction of H2 with the active site; the alternative mechanism shown in Scheme 2 represents a feasible path instead, according to the overall reaction energy profile here reported. Only after one-electron reduction and protonation of the active site and water dissociation, Ni−B is converted into the active Ni−SIa state, which is able to bind a H2 molecule and start physiologic catalysis in the oxidative reaction. Activation of Ni−A. As already mentioned, this study is the first computational investigation of the activation mechanism of Ni−A in which the latter is modeled as a species containing a hydroxide ligand bridging the NiIIIFeII site and the sulfur atom of the Cys64 residue oxidized to bridging sulfenate as recently suggested by a crystallographic structure that was confidently associated to the Ni−A state.49 The good agreement between our predicted Ni−A structure and X-ray data has been discussed in a previous work on the same active site model.50 Main results obtained from the current study on the Ni−A reactivation mechanism are schematically represented in Scheme 3. Analogous to the case of Ni−B, the monoelectronic reduction and the protonation of the active site are investigated as first steps of the activation process. This choice is due to the fact that the pH dependence of the midpoint potential of the Ni−A/Ni−SU couple suggests that the one-electron reduction of Ni−A required for the formation of the diamagnetic Ni−SU state80,86 is accompanied by a single proton transfer step.87 In particular, proton transfer from the Glu14 residue to the sulfur atom of Cys555 and subsequently to the OH− ligand is investigated for Ni−A and for its one-electron reduced state. However, the Ni−A species in which the sulfur atom of Cys555 is protonated cannot be isolated as a stable intermediate since during geometry optimization it converges to the reactant with the spontaneous migration of the hydrogen atom to the nearby carboxylate group of Glu14. Conversely, the corresponding monoreduced state (1O2H2(−4,1)) is a stable species, even though it is less stable than reactant 1-O2HGluH(−4,1) by 2.7 kcal/mol (see Scheme 3 and Figure 4). Intermediates in which the proton is transferred to the bridging oxygenic ligand, with consequent formation of a water molecule, are instead stable species in both redox states (2-O2H2(−3,2) and 2-O2H2(−4,1) in Scheme 3). However, 2O2H2(−3,2) is less stable than corresponding isomer 1O2HGluH(−3,2) by 15.9 kcal/mol, clearly indicating that the transfer of a hydrogen atom from the Glu14 residue to the hydroxide ligand of Ni−A is strongly unfavorable when the Ni atom is in the NiIII redox state. Reduction of the active site promotes the proton transfer from Cys555 to the bridging OH− ligand, as in this case the process is slightly exoenergetic (−2.3 kcal/mol), and it is characterized by an activation energy equal to about 10 kcal/mol, a value similar to that calculated for the corresponding process for Ni−B (see Scheme 3 and Figure S1). The possibility that protonation occurs at the oxygen atom of the bridging sulfenate bound to the sulfur atom of Cys64 was also considered. Nevertheless, the adduct featuring protonation of this atom is less stable than 1O2H2(−4,1) by 9.5 kcal/mol.

Scheme 4. Coordination Geometry of the Ni Atom for Selected Species Involved in the Reactivation Mechanism of the (a) Ni−B and the (b) Ni−A States

In a previous work, we have discussed the crucial role of the Sa−Ni−Sb angle for the binding and activation of H2 in the Ni−SIa state of the enzyme.62 The optimal value of this angle for H2 binding has been evaluated to be about 120° corresponding to a peculiar seesaw geometry of the Ni atom, which in the enzyme is stabilized by the protein environment.62 Increasing this angle affects the electronic structure of the bimetallic cluster by reducing the ability of Ni and concomitantly by increasing the ability of Fe to bind H2. In fact, we observe that such effect is extended to the binding of H2O to NiIIFeII forms of the enzyme, and it is modulated by the presence of the sulfenate oxygen atom in Ni−A species (see Scheme 4). In the H2O-adduct of Ni−B (2-OH2(−4,1)), the value of the Sa−Ni−Sb angle is equal to about 110°, and terminal coordination of H2O to the Ni atom is preferred. In the corresponding Ni−A species (2-O2H2(−4,1)), the Sa−Ni−Sb angle is more than 30° larger and H2O binds terminally to the Fe atom. To confirm this observation, we can note that the isomer of 2-O2H2(−4,1) in which the water molecule is terminally coordinated to Ni has been identified as a genuine energy minimum, but it is less stable than 2-O2H2(−4,1) by 10.3 kcal/mol. H


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Inorganic Chemistry

the active site of this species are investigated as described in the following lines. According to the catalytic mechanism, previously described for [NiFe]-hydrogenases,62 the oxidative addition of H2 on the Ni−SIa state occurs at the Ni atom, and it is followed by H2 cleavage and transfer of a proton from Ni to the sulfur atom of the terminally coordinated Cys555 residue and then to the carboxylate group of Glu14. Subsequently, two electrons and two protons are transferred from the active site to reform the Ni−SIa state. Considering 1-O(−4,1), binding and activation of H2 may follow a similar pathway. However, the geometry of the Ni atom in 1-O(−4,1) adopts a more square planar configuration with respect to the seesaw geometry of Ni−SIa, with the Sa−Ni−Sb and Sc−Ni−OSd angles equal to about 140 and 169°, respectively, compared to the corresponding values of 124 and 170° calculated for Ni−SIa. Inspection of frontier orbitals reveals that this conformational change has a significant impact on the electronic structure of the bimetallic complex by reducing the contribution of Ni to the LUMO (which is the principal orbital involved in the interaction with H2) and concomitantly increasing the contribution of the Fe atom (see Figure S4). Therefore, we may expect that in 1O(−4,1) H2 binding preferentially occurs on the Fe atom with respect to Ni. In fact, H2 can coordinate both metal atoms with the binding to Fe (−1.8 kcal/mol) to give 3-OH2(−4,1) (see Scheme 3), slightly more favorable than the binding to Ni (−1.3 kcal/mol), the latter yielding 4-OH2(−4,1). Nevertheless, in both cases H2 binding is less favorable than the corresponding process for Ni−SIa (−4.4 kcal/mol). Analogously to the case of the Ni−SIa−H2 adduct, in 4OH2(−4,1) the H2 molecule binds side-on to the Ni atom, with the averaged Ni−H bond distance equal to 1.67 Å and the Fe− Ni distance of 2.85 Å (see Figure 4). H2 also coordinates to the Fe atom in 3-OH2(−4,1) in a side-on mode. In this case, the H2 molecule binds trans to the CO ligand completing the octahedral coordination of the Fe atom, and one of the two hydrogen atoms occupies a semibridging position between the two metals (see Figure 4). The averaged Fe−H bond distance and the Fe−Ni distance are equal to 1.75 and 3.32 Å, respectively, whereas the Sa−Ni−Sb angle is as large as 149°. Since binding of H2 to both Ni and Fe yields almost isoenergetic species, in the following we will consider the heterolytic splitting of H2 for both isomers. The H2 cleavage step in 4-OH2(−4,1) to give intermediate 5-OH2(−4,1), featuring one terminal and one bridged H atom, is a slightly endoenergetic process (+2.7 kcal/mol), and it is characterized by a very small activation energy equal to about 3 kcal/mol (transition state shown in Figure S2). The following proton transfer from Ni to the sulfur atom of Cys555 leading to 6OH2(−4,1) complex and then to the carboxyl group of Glu14 leading to 2-OHGluH(−4,1) are exoenergetic by 10.9 and 1.9 kcal/mol, respectively, with the former step being almost barrierless (see Scheme 3). Interestingly, the heterolytic cleavage of H2 bound to the Fe atom in 3-OH2(−4,1), to give a bridging and a terminal hydride coordinated to the Fe atom is not a spontaneous process as the intermediate with the two separated H atoms converged to the H2 adduct species. The cleavage of H2 on Fe can then be performed by the migration of the semibridging hydrogen atom to either the terminal position on Ni to give 5-OH2(−4,1), or directly to the sulfur atom of the Cys555 residue to give 6OH2(−4,1). The energy differences associated to these two steps (+3.3 and −7.6 kcal/mol, respectively) allow us to suppose

As in the case of Ni−B, several positions near the active site have been considered for the H2O molecule as product of the dissociation process (see Figure S2). Interestingly, the first energy minimum along the dissociation coordinate (2+O2H2A(−4,1)) has been found to have a position of H2O very similar to the one calculated for the corresponding Ni−B species, which is more than 5 Å far from the Fe atom (see Figure S3). This intermediate is 7.5 and 7.0 kcal/mol less stable than 2O2H2(−4,1) reactant using the large and the medium models, respectively. In addition, the activation energy for the dissociation of H2O from 2-O2H2(−4,1) is equal to 11.5 kcal/ mol, a value significantly higher than that calculated for the corresponding Ni−B species. It is also interesting to note that in the transition state structure the Fe- - -OH2 distance is as long as 3.6 Å (see Figure 3b). Migration of H2O from 2+O2H2-A(−4,1) to the nearby positions already discussed for the Ni−B species are exoenergetic processes; indeed, the intermediates in which the H2O molecule is H-bonded to the carboxylic oxygen atom of Asp103 and the carbonyl oxygen of Val63 (2+O2H2-B(−4,1)) and in which H2O is H-bonded to Cys61, Cys64, and His68 (2+OH2-C(−4,1)) are more stable than 2+O2H2-A(−4,1) by 10.3 and 6.9 kcal/mol, respectively. In summary, the results presented above indicate that as observed for Ni−B, monoelectronic reduction of Ni−A promotes protonation of the bridging ligand. This protonation step is significantly less exoenergetic for Ni−A compared to Ni−B, even if activation energies are similar. The formed H2O molecule results to be terminally coordinated on the two different metal centers: the Ni atom for Ni−B and the Fe atom for Ni−A. Dissociation of H2O from the Ni−A derivative 2O2H2(−4,1) is much less favorable than the corresponding process for Ni−B, due to both the significantly larger energy barrier and the higher energy of the intermediate formed along the dissociation coordinate. This result supports the hypothesis that the rate determining step of the Ni−A activation process is the release of the water molecule. The removal of the bridging oxygenic species during the Ni−A reductive activation is confirmed by 17O-labeling experiments.88 These results in conjunction with experimental evidence allow us to propose a structure for the active site of [NiFe]hydrogenases in the Ni−SU state. Experiments show that the Ni−A to Ni−SU conversion consists in a fast and reversible one-electron/one-proton step,52,89,90 and that the rate-limiting step of the activation process is the conversion of Ni−SU to the Ni−SIa active state. The diamagnetic Ni−SU state should therefore correspond to the monoreduced species, 2O2H2(−4,1), in which the bridging OH− ligand is protonated to give a H2O molecule terminally coordinated to the Fe atom. The rate-determining step, independent of redox potential and pH,35,87 is the endoenergetic migration of this H2O molecule to a position nearby the active site. The removal of H2O from the active site leads to 1-O(−4,1) which corresponds to the Ni−SIa state, but with the sulfenate oxygen atom still inserted between the Ni and the sulfur atom of Cys61 (see Scheme 3 and Figure 4). Analogous to Ni−SIa, we found that the “low-spin” diamagnetic NiII species is more stable than the “high-spin” triplet one (1-O(−4,3)) by about 8 kcal/mol. In 1-O(−4,1) , the two metal ions are both characterized by one vacant coordination position, where H2 might bind as in the Ni−SIa state. Therefore, with the aim of exploring the catalytic properties of 1-O(−4,1) and comparing them with those of Ni−SIa, dihydrogen binding and cleavage at I


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Inorganic Chemistry

Scheme 5. Schematic Representation of Electron and Proton Transfers Required for the Release of the Oxygen Atom of the Sulfenate Group as Water Molecule from the 1-O(−4,1) Active Site in the Absence of H2a

a 9-OH2(−4,1) can convert to the Ni−SIa state through the release of a water molecule as shown in Scheme 3. The most plausible mechanism is indicated by bold arrows. For those species in which Glu14 is not explicitly drawn, it is modeled as glutamate. Energy differences are in kcal/mol.

been considered. One reaction pathway implies the transfer of the hydrogen atom terminally coordinated to Ni in 5-OH2(−4,1) directly to the oxygen atom of the sulfenate group. This process, yielding 7-OH2(−4,1), is favored by 5.5 kcal/mol, and it is characterized by an energy barrier of 13.5 kcal/mol (see Figure S2 for transition state). An alternative pathway involves the conversion of 5-OH2(−4,1) to 6-OH2(−4,1) and then to 2OHGluH(−4,1). As already mentioned, the transfer of the hydrogen atom terminally coordinated to Ni to the Glu14 residue is exoenergenetic by 12.8 kcal/mol. After this process, the hydride bridging the two metal atoms can be transferred to the oxygen atom of the sulfenate group (2-OHGluH(−4,1) → 3OHGluH(−4,1)). As shown in Scheme 3, this process is exoenergetic by 11.0 kcal/mol. Instead, if either the bridging hydride ligand or the hydrogen atom bound to the sulfur atom of Cys555 in 6-OH2(−4,1) are directly transferred to the sulfenate oxygen atom (6-OH2(−4,1) → 8-OH2(−4,1) or 6OH2(−4,1) → 7-OH2(−4,1), respectively), then the protonation step is endoenergetic by 21.6 or 5.4 kcal/mol. It should be noted that protonation of the glutamate residue near the active site, not only promotes the protonation of the sulfenate group, but also the cleavage of the SCys64−O bond, as indicated by the increase of the SCys64−O distance from 1.66 Å in 6-OH2(−4,1) to 3.02 Å in 3-OHGluH(−4,1). As a consequence, the Ni−Fe distance also increases from 2.74 Å in 5-OH2(−4,1) to 3.87 in 3-OHGluH(−4,1) (see Figure S3) We have also investigated the direct transfer of the peripheral hydrogen atom of H2 coordinated to the Fe atom in 3-OH2(−4,1) to the sulfenate oxygen atom to give 7-OH2(−4,1). This process is slightly exoenergetic (−2.3 kcal/mol), suggesting that this is a reliable pathway, but we were not able to identify a transition state connecting these two intermediates. Second protonation of the sulfenate group can be thus achieved by the transfer of the bridging hydrogen atom in 7OH2(−4,1) or of the hydrogen atom coordinated to the Glu14 residue in 3-OHGluH(−4,1) to the Ni−O(H)−SCys64 sulfenic group. These processes, yielding 9-OH2(−4,1), are exoenergetic

that the heterolytic cleavage of H2 on Fe leads to intermediate 6-OH2(−4,1). Notably, the energy barrier associated to this process is 9.2 kcal/mol (see Figure S2 for transition state). 6OH2(−4,1) can further evolve to 2-OHGluH(−4,1), already discussed for the reaction of H2 on the Ni atom (see Scheme 3). The formation of the one-electron-oxidized paramagnetic intermediate 2-OH(−4,2) from 2-OHGluH(−4,1), corresponding to the Ni−C state in the catalytic mechanism, is predicted to be disfavored by the presence of the bridging sulfenate. This is suggested by comparison of the energy of HOMO in 2OHGluH(−4,1) (−0.96 eV) and in the corresponding catalytic species Ni−R (−0.69 eV). This significant energy difference indicates that oxidation of 2-OHGluH(−4,1) requires more positive potentials than those needed for oxidation of Ni−R, suggesting that the presence of an O atom coordinated to Ni makes the electron transfer from the active site unfavorable, thus preventing the formation of the paramagnetic species, 2OH(−4,2). The overall picture coming from our calculations is compatible with EPR experiments that until now have detected only four EPR-active states of the [NiFe]-hydrogenases active site: Ni−A, Ni−B, Ni−C, and Ni−L. Reduction of the oxygenic species trapped between the Ni and the S atom of Cys64 is therefore necessary to promote formation of an active intermediate of the enzyme. Thus, the activation of the Ni−SU state requires further reducing equivalents that should be simultaneously available in order to avoid the formation of paramagnetic states, whose occurrence was actually not experimentally observed. These electrons might be readily supplied by the oxidation of an H2 molecule that, after its binding to the active site in 1-O(−4,1) (vide supra), can also provide the H atoms required for the release of the trapped oxygen atom as water molecule. In line with this hypothesis, the overall reaction 4-OH2(−4,1) → 9OH2(−4,1) is calculated to be energetically favored by about 30 kcal/mol. Different pathways for the first protonation of the oxygen atom of the sulfenate group, after H2 cleavage, have J


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Inorganic Chemistry

study is the first computational investigation of [NiFe]hydrogenases activation mechanism describing the Ni−A state as containing a bridging hydroxide ligand and the Cys64 residue oxidized to bridging sulfenate. A different number of reducing equivalents are required to reactivate Ni−B and Ni−A. According to our proposed mechanism for [NiFe]-hydrogenases activation, the conversion of Ni−B to Ni−SIa is achieved by one-electron reduction and protonation of the active site, which promote the removal of the bridging hydroxide ligand as a water molecule (NiIII(OH−)FeII + e− + H+ → NiIIFeII + H2O). Two further electrons are required to reduce the sulfur atom of the sulfenate group present in the Ni−A state. These two electrons are provided by a H2 molecule that through the binding to the active site also provides the two hydrogen atoms required for the release of the sulfenate oxygen as a water molecule (NiIII(OH−)(OCys64)FeII + e− + H+ + H2 → NiIIFeII + 2H2O) according to the mechanism described in the “Results” section. The hypothesis that a H2 molecule provides two of the three electrons required for Ni−A activation is supported by the fact that two reducing equivalents must be simultaneously available, since no paramagnetic species have been detected during the activation process. Furthermore, activation of [NiFe]-hydrogenases is always achieved in the presence of H2 or using medium containing low-potential electron donors that can generate H2.81,87,91−96 The possibility that two electrons are simultaneously supplied by external reductants is ruled out by dynamic electrochemical kinetic studies showing that in the absence of H2 electrons alone are not able to reactivate Ni−A; only a slow reversible equilibrium between Ni−A and an intermediate state that is still not active can be established.35 H2 is required to make this reductive transformation irreversible and to convert this intermediate to active enzyme. Therefore, both electrons and H2 separately are required to activate the Ni−A state as revealed by protein film voltammetry which allowed to resolve electron and chemical events in the time and potential dimensions. Although characterized by a different energetic profile, the first steps of the Ni−A activation process are the same as those required to reactivate Ni−B: (i) one-electron reduction, (ii) protonation of the bridging hydroxide ligand, and (iii) release of a water molecule. A comparison of the two reaction profiles explains the different reactivation kinetic of Ni−B and Ni−A. To study the reduction propensity of the active site in the Ni− B and Ni−A states, the LUMO energies of these species are analyzed. Interestingly, they are almost identical (−1.31 eV for Ni−B and −1.29 eV for Ni−A). This observation suggests that the same reducing conditions are required for the first oneelectron reduction of the active site. Reaction energies associated to the transfer of a proton from the Glu14 residue to the hydroxide ligand indicate that the OH− protonation is significantly exoenergetic process for the monoreduced species of Ni−B (−9.6 kcal/mol) whereas it is slightly endoenergetic for the monoreduced species of Ni−A (+0.4 kcal/mol) although both process are characterized by similar activation energies in the proton transfer from the sulfur atom of Cys555 to the hydroxy ligand (8.5 and 9.6 kcal/ mol). At this point, the sulfenate oxygen atom clearly affect the stereoelectronic features of the bimetallic cluster governing the regiochemistry of H2O and H2 binding. Indeed, in the Ni−B state, H2O spontaneously moves from the bridging position to the terminal coordination site of the Ni atom, whereas in the case of Ni−A, H2O moves to a terminal position on the Fe

by 27.1 and 8.8 kcal/mol, respectively. In this species (see Figure S3), the SCys64−O distance decreases from 3.02 to 2.86 Å, and the water molecule is terminally coordinated to the Ni atom (Ni−O distance = 1.99 Å), which features and almost ideal square planar geometry. Dissociation of H2O from 9OH2(−4,1) leads to the shortening of the Ni−SCys64 distance from 3.83 to 2.26 Å and the consequent formation of the active Ni−SIa state (see Scheme 3). It is worth noting that water release from the active site is a very exoenergetic process, considering all the adjacent positions for the H2O molecule. In particular, 2+OH2-A(−4,1), 2+OH2-B (−4,1), and 2+OH2-C(−4,1) are more stable than 9-OH2(−4,1) by 25.3, 35.2, and 31.9 kcal/ mol, respectively. The possibility that the two electrons required for the full reduction of the sulfenate group are supplied by external reducing agents, and not by a H2 molecule, was also investigated. As shown in Scheme 5, 1-O(−4,1) can receive two electrons and two protons to give, independently from the order in which they arrive at the active site, the 9-OH2(−4,1) complex, that further evolves to the Ni−SIa state through the release of the trapped oxygen atom as water molecule. Notably, the proton transfer from the Glu14 residue to the O atom of the sulfenate group of 1-O(−4,1) is strongly disfavored (1OGluH(−3,1) → 3-OH(−3,1), +14.5 kcal/mol). Conversely, this process is exoenergetic by 4.9 and 37.1 kcal/mol for the mono(1-O(−5,2)) and di- (1-O(−6,1)) reduced species of 1-O(−4,1), respectively. These results suggest that the reduction of the active site promotes the protonation of the oxygen atom of the Ni−O−SCys64 sulfenate group. Analogously, protonation of the resulting Ni−O(H)−SCys64 sulfenic group is promoted by reducing the active site. However, in this case, the reaction is energetically favored only for the two-electrons reduced species (3-OHGluH(−4,1) → 9-OH2(−4,1), −8.8 kcal/mol). On the basis of these results and considering that no detectable paramagnetic species are formed during the activation process, we can assume that in the absence of H2 the trapped oxygen atom in 1-O(−4,1) is removed from the active site by a twoelectron reduction followed by double protonation of the active site. However, this possibility can be ruled out; extreme reducing conditions are required for this process. Furthermore, two successive one-electron reductions, each of which accompanied by a single proton transfer step, are more favored than a two-electrons/two-protons step. Protoncoupled electron transfers are in fact very common reactions in chemistry and biology to balance the charge of the system. Furthermore, dynamic electrochemical kinetic studies showed that, in the absence of H2, electrons alone are not able to reactivate Ni−A.35

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DISCUSSION [NiFe]-hydrogenases are enzymes of considerable interest for their potential employment in H2 production processes in the presence of ambient O2 levels. For this reason, the reactivation pathway of the inactive and oxidized active site of these enzymes is a crucial issue. In this context, the goal of this computational study is to elucidate the reactivation mechanism of the oxidized and inactive Ni−B and Ni−A states. To do this, a very large DFT model of the active site and its protein environment has been used. It is worth noting that confidence with which the occurrence of certain enzyme forms is discussed in the present study is due the reliability of the large-size model of the enzyme which provides high quality reaction energy profiles.50,62,63 It should also be noted that this K


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Inorganic Chemistry

insights into the reactivation mechanisms. In particular, the rate-determining step of the reactivation process is shown to be different for Ni−B and Ni−A. One-electron reduction of the active site is proposed to limit the reactivation rate of Ni−B. On the other hand, the rate-determining step of the Ni−A reactivation is the release of the water molecule coordinated to the Fe atom, which is formed after one-electron reduction of Ni−A and protonation of the bridging hydroxide ligand.

atom. This effect can be explained by considering the subtle changes of the geometry of the Ni atom induced by the coordinated sulfenate oxygen atom. In one-electron-reduced forms of Ni−B, the NiII atom when tetracoordinated features an almost ideal seesaw coordination geometry, whereas in the one-electron-reduced forms of Ni−A the NiII atom adopts a more square planar conformation. The effects of this conformational modification are evident by inspecting the frontier orbitals of the two species. In the Ni−B derivative, the LUMO is more localized on the Ni atom, whereas in the Ni−A derivative, it is more localized on the Fe atom. The result of these stereoelectronic differences is that the energy barrier for the dissociation of H2O is significantly higher in Ni−A with respect to Ni−B. In addition, the first energy minimum identified along the dissociation coordinate is significantly less stable than the H2O-adduct in the case of Ni−A. Based on these considerations, it is possible to suggest that the slower activation of Ni−A compared to that of Ni−B is due to the different energetic requirements for the dissociation of H2O from the H2O adducts. The slow step of the Ni−B activation is here proposed to precede formation and release of H2O, and corresponds to the one-electron reduction of Ni−B. The fact that the Ni−B to Ni−SIa conversion is rate-limited by the redox potential supports this hypothesis.97 On the other hand, the dissociation of H2O from the H2O-adduct is proposed to be the rate-determining step of the Ni−A activation process, independent of redox potential and pH.35,87 According to this hypothesis, dynamic electrochemical kinetic studies established that fast and reversible electron transfer precedes the ratedetermining step which is followed by a reaction with H2 that completes the reductive activation process.35 The energetic feasibility of the H2 binding to the active site and of the subsequent transfers of the two hydrogen atoms to the sulfenate group is confirmed by the fact that the activation rate constant is independent of the partial pressure of hydrogen.94 Notably, our results also allow us to confidently propose reasonable structures for the inactive Ni−SIr and Ni−SU states, formed respectively from the one-electron reduction of Ni−B and Ni−A. Two forms for the Ni−SIr state in which an oxygenic ligand is still bound to the active site have been identified. The difference between these two structures lies in the protonation state of such ligand, as suggested by spectroelectrochemical experiments,52 and in its coordination mode: at high pH values, a hydroxide bridges the Ni and Fe atoms, whereas at lower pH values, a H2O molecule terminally binds the Ni atom (1-OH(−5,1) and 2-OH2(−4,1), respectively, in Scheme 2). In contrast, FTIR-spectroelectrochemical and electrochemical data showed that Ni−A is converted to Ni− SU in a fast and reversible electrochemical step involving one electron and one proton, while the conversion of Ni−SU to Ni−SIa, independent of the redox potential, is the ratedetermining step of the overall reactivation process.35,81,87,91,92 On the basis of these considerations, the Ni−SU state is proposed to be 2-O2H2(−4,1) (see Scheme 3), in which a H2O molecule is coordinated to the Fe atom and the Cys64 residue is oxidized to sulfenate. In summary, our results provide a better understanding of the origin of the different reactivation kinetics of the oxidized and inactive Ni−B and Ni−A states of [NiFe]-hydrogenases by revealing the subtle effect played by the sulfenate oxygen atom of Ni−A in switching the regiochemistry of the H2O and H2 binding from the Ni to the Fe atom, through the modification of the coordination environment of Ni. This leads to significant

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02348.

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Detailed information list of the atoms composing the active site model, list of the selected atoms that, during geometry optimizations, have been constrained to the crystallographic positions to avoid unrealistic distortions at the boundary of the model, structural details and electronic structure properties of selected species, and energies of all species investigated in this work (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +39-0264482816. Fax: +39-0264482890. ORCID

Claudio Greco: 0000-0001-9628-7875 Maurizio Bruschi: 0000-0002-5709-818X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge CINECA for the availability of highperformance computing resources as part of the agreement with the University of Milano-Bicocca.

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REFERENCES

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