Probing the effects of one-electron reduction and protonation on the electronic properties of the Fe-S clusters in the active-ready form of [FeFe]-hydrogenases. A QM/MM investigation

A QM/MM investigation of the active-ready (H(ox)) form of [FeFe]-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H(2) formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in H(ox) is unlikely, and in any case it would not trigger electron transfer from the accessory Fe(4)S(4) clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed.     

Structural and electronic properties of the [FeFe] hydrogenase H-cluster in different redox and protonation states. A DFT investigation

The molecular and electronic structure of the Fe 6S 6 H-cluster of [FeFe] hydrogenase in relevant redox and protonation states have been investigated by DFT. The calculations have been carried out according to the broken symmetry approach and considering different environmental conditions. The large negative charge of the H-cluster leads, in a vacuum, to structures different from those observed experimentally in the protein. A better agreement with experimental data is observed for solvated complexes, suggesting that the protein environment could buffer the large negative charge of the H-cluster. The comparison of Fe 6S 6 and Fe 2S 2 DFT models shows that the presence of the Fe 4S 4 moiety does not affect appreciably the geometry of the [2Fe] H cluster. In particular, the Fe 4S 4 cluster alone cannot be invoked to explain the stabilization of the mu-CO forms observed in the enzyme (relative to all-terminal CO species). As for protonation of the hydrogen cluster, it turned out that mu-H species are always more stable than terminal hydride isomers, leading to the conclusion that specific interactions of the H-cluster with the environment, not considered in our calculations, would be necessary to reverse the stability order of mu-H and terminal hydrides. Otherwise, protonation of the metal center and H 2 evolution in the enzyme are predicted to be kinetically controlled processes. Finally, subtle modifications in the H-cluster environment can change the relative stability of key frontier orbitals, triggering electron transfer between the Fe 4S 4 and the Fe 2S 2 moieties forming the H-cluster.     

Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.     

Structural insights into the active-ready form of [FeFe]-hydrogenase and mechanistic details of its inhibition by carbon monoxide

[FeFe]-hydrogenases harbor a {2Fe3S} assembly bearing two CO and two CN- groups, a mu-CO ligand, and a vacant coordination site trans to the mu-CO group. Recent theoretical results obtained studying the isolated {2Fe3S} subsite indicated that one of the CN- ligands can easily move from the crystallographic position to the coordination site trans to the mu-CO group; such an isomerization would have a major impact on substrates and inhibitors binding regiochemistry and, consequently, on the catalytic mechanism. To shed light on this crucial issue, we have carried out hybrid QM/MM and free energy perturbation calculations on the whole enzyme, which demonstrate that the protein environment plays a crucial role and maintains the CN- group fixed in the position observed in the crystal structure; these results strongly support the hypothesis that the vacant coordination site trans to the mu-CO group has a crucial functional relevance both in the context of CO-mediated inhibition of the enzyme and in dihydrogen oxidation/evolution catalysis.     

A theoretical study on the enhancement of functionally relevant electron transfers in biomimetic models of [FeFe]-hydrogenases

Recent advances aimed at modeling the chemistry of the active site of [FeFe]-hydrogenases (the H-cluster, composed by a catalytic Fe(2)S(2) subcluster and an Fe(4)S(4) portion) have led to the synthesis of binuclear coordination compounds containing a noninnocent organophosphine ligand [2,3-bis(diphenylphosphino)maleic anhydride, bma] that is able to undergo monoelectron reduction, analogously to the tetranuclear Fe(4)S(4) subcluster portion of the H-cluster. However, such a synthetic model was shown to feature negligible electronic communication between the noninnocent ligand and the remaining portion of the cluster, at variance with the enzyme active site. Here, we report a theoretical investigation that shows why the electron transfer observed in the enzyme upon protonation of the catalytic Fe(2)S(2) subsite cannot take place in the bma-containing cluster. In addition, we show that targeted modifications of the bma ligand are sufficient to restore the electronic communication within the model, such that electron density can be more easily withdrawn from the noninnocent ligand, as a result of protonation of the iron centers. Similar results were also obtained with a ligand derived from cobaltocene. The relevance of our findings is discussed from the perspective of biomimetic reproduction of proton reduction to yield molecular hydrogen.     

DFT investigation of structural, electronic, and catalytic properties of diiron complexes related to the [2Fe](H) subcluster of Fe-only hydrogenases

Hydrogenases catalyze the reversible oxidation of dihydrogen to protons and electrons. The structures of two Fe-only hydrogenases have been recently reported [Peters, J. W.; Lanzilotta, W. N.; Lemon, B. J.; Seefeldt, L. C. Science 1998, 282, 1853-1858. Nicolet, Y.; Piras, C.; Legrand, P.; Hatchikian, E. C.; Fontecilla-Camps, J. C. Structure 1999, 7, 13-23], showing that the likely site of dihydrogen activation is the so-called [2Fe](H) cluster, where each Fe ion is coordinated by CO and CN(-) ligands and the two metals are bridged by a chelating S-X(3)-S ligand. Moreover, the presence of a water molecule coordinated to the distal Fe2 center suggested that the Fe2 atom could be a suitable site for binding and activation of H(2). In this contribution, we report a density functional theory investigation of the structural and electronic properties of complexes derived from the [(CO)(CH(3)S)(CN)Fe(II)(mu-PDT)Fe(II)(CO)(2)(CN)](-1) species, which is related to the [2Fe](H) cluster observed in Fe-only hydrogenases. Our results show that the structure of the [2Fe](H) cluster observed in the enzyme does not correspond to a stable form of the isolated cluster, in the absence of the protein. As a consequence, the reactivity of [(CO)(CH(3)S)(CN)Fe(II)(mu-PDT)Fe(II)(CO)(2)(CN)](-1) derivatives in solution may be expected to be quite different from that of the active site of Fe-only hydrogenases. In fact, the most favorable path for H(2) activation involves the two metal atoms and one of the bridging S atoms and is associated with a very low activation energy (5.3 kcal mol(-1)). The relevance of these observations for the catalytic properties of Fe-only hydrogenases is discussed in light of available experimental and theoretical data.     

Regioselectivity of H cluster oxidation

The H(2)-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O(2)-induced structural changes in the [Fe(4)S(4)] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O(2)(-) and HO(2)(-). After protonation of O(2)(-), an OOH radical may coordinate to the Fe atoms of the cubane, whereas H(2)O(2) specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O(2)-induced inhibition is accompanied by distortions of the [Fe(4)S(4)] moiety and account for the irreversibility of this process.     

Biosynthesis of the catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE,HydF, and HydG

[FeFe] hydrogenases carry out the redox interconversion of protons and molecular hydrogen (2H⁺ + 2e⁻ ⇌ H₂) at a complex Fe–S active site known as the H-cluster. The H-cluster consists of a [4Fe–4S] subcluster, denoted here as [4Fe]_H, linked via a cysteine sulfur to an interesting organometallic [2Fe]_H subcluster thought to be the subsite where the catalysis occurs. This [2Fe]_H subcluster consists of two Fe atoms, linked with a bridging CO and a bridging SCH₂NHCH₂S azadithiolate (adt), with additional terminal CO and CN ligands bound to each Fe. Synthesizing such a complex organometallic unit is a fascinating problem in biochemistry, complicated by the toxic nature of both the CO and CN⁻ species and the relative fragility of the azadithiolate bridge. It has been known for a number of years that this complex biosynthesis is carried out by a set of three essential Fe–S proteins, HydE, HydF, and HydG. HydF is a GTPase, while HydE and HydG are both members of the large family of radical S-adenosylmethionine (rSAM) enzymes. In this perspective we describe the history of research and discovery concerning these three Fe–S “maturase” proteins and describe recent evidence for a sequential biosynthetic pathway beginning with the synthesis of a mononuclear organometallic [Fe(ii)(CO)₂CN(cysteine)] complex by the rSAM enzyme HydG and its subsequent activation by the second rSAM enzyme HydE to form a highly reactive Fe(i)(CO)₂(CN)S species. In our model a pair of these Fe(i)(CO)₂(CN)S units condense to form the [Fe(CO)₂(CN)S]₂ diamond core of the [2Fe]_H cluster, requiring only the installation of the central CH₂NHCH₂ portion of the azadithiolate bridge, whose atoms are all sourced from the amino acid serine. This final step likely occurs with an interplay of HydE and HydF, the details of which yet remain to be elucidated.

Fe–S cluster enzymes HydG, HydE, and HydF provide sequential assembly of the catalytic H-cluster of [FeFe] hydrogenase.     

Peptide hydrolysis catalyzed by matrix metalloproteinase 2: a computational study

The MMP-2 reaction mechanism is investigated by using different computational methodologies. First, quantum mechanical (QM) calculations are carried out on a cluster model of the active site bound to an Ace-Gly approximately Ile-Nme peptide. Along the QM reaction path, a Zn-bound water molecule attacks the Gly carbonyl group to give a tetrahedral intermediate. The breaking of the C-N bond is completed thanks to the Glu 404 residue that shuttles a proton from the water molecule to Ile-N atom. The gas-phase QM energy barrier is quite low ( approximately 14 kcal/mol), thus suggesting that the essential catalytic machinery is included in the cluster model. A similar reaction path occurs in the MMP-2 catalytic domain bound to an octapeptide substrate according to hybrid QM and molecular mechanical (QM/MM) geometry optimizations. However, the rupture of the Gly( P 1) approximately Ile( P 1') amide bond is destabilized in the static QM/MM calculations, owing to the positioning of the Ile( P 1') side chain inside the MMP-2 S 1' pocket and to the inability of simple energy miminization methodologies to properly relax complex systems. Molecular dynamics simulations show that these steric limitations are overcome easily through structural fluctuations. The energetic effect of structural fluctuations is taken into account by combining QM energies with average MM Poisson-Boltzmann free energies, resulting in a total free energy barrier of 14.8 kcal/mol in good agreement with experimental data. The rate-determining event in the MMP-2 mechanism corresponds to a H-bond rearrangement involving the Glu 404 residue and/or the Glu 404-COOH --> N-Ile( P 1') proton transfer. Overall, the present computational results and previous experimental data complement each other well in order to provide a detailed view of the MMPs catalytic mechanism.     

Isocyanide in Biochemistry? A Theoretical Investigation of the Electronic Effects and Energetics of Cyanide Ligand Protonation in [FeFe]‐Hydrogenases

The presence of Fe‐bound cyanide ligands in the active site of the proton‐reducing enzymes [FeFe]‐hydrogenases has led to the hypothesis that such Brønsted–Lowry bases could be protonated during the catalytic cycle, thus implying that hydrogen isocyanide (HNC) might have a relevant role in such crucial microbial metabolic paths. We present a hybrid quantum mechanical/molecular mechanical (QM/MM) study of the energetics of CN^? protonation in the enzyme, and of the effects that cyanide protonation can have on [FeFe]‐hydrogenase active sites. A detailed analysis of the electronic properties of the models and of the energy profile associated with H₂ evolution clearly shows that such protonation is dysfunctional for the catalytic process. However, the inclusion of the protein matrix surrounding the active site in our QM/MM models allowed us to demonstrate that the amino acid environment was finely selected through evolution, specifically to lower the Brønsted–Lowry basicity of the cyanide ligands. In fact, the conserved hydrogen‐bonding network formed by these ligands and the neighboring amino acid residues is able to impede CN^? protonation, as shown by the fact that the isocyanide forms of [FeFe]‐hydrogenases do not correspond to stationary points on the enzyme QM/MM potential‐energy surface.     

Modeling [Fe-Fe] hydrogenase: evidence for bridging carbonyl and distal iron coordination vacancy in an electrocatalytically competent proton reduction by an iron thiolate assembly that operates through Fe(0)-Fe(II) levels

IR spectroelectrochemistry of Fe4{Me(CH2S)3}2(CO)8 (4Fe6S) in the nu(CO) region shows that the neutral and anion forms have all their CO groups terminally bound to the Fe atoms; however, for the dianion there is a switch of the coordination mode of at least one of the CO groups. The available structural and nu(CO) spectra are closely reproduced by density-functional theory calculations. The calculated structure of 4Fe6S2- closely mirrors that of the diiron subsite of the [Fe-Fe] hydrogenase H cluster with a bridging CO group and an open coordination site on the outer Fe atom of pairs of dithiolate-bridged Fe0FeII subunits connected by two bridging thiolates. Geometry optimization based on the all-terminal CO isomer of 4Fe6S2- does not give a stable structure but reveals a second-order saddle point ca. 11.53 kcal mol(-1) higher in energy than the CO-bridged form. Spectroelectrochemical studies of electrocatalytic proton reduction by 4Fe6S show that slow turnover from the primary reduction process (E1/2'=-0.71 V vs Ag/AgCl) involves rate-limiting protonation of 4Fe6S- followed by reduction to H:4Fe6S-. Rapid electrocatalytic proton reduction is obtained at potentials sufficient to access 4Fe6S2-, where the rate of dihydrogen elimination from the FeIIFeII core of 4Fe6S is ca. 500 times faster than that from the FeIFeI core of Fe2(mu-S(CH2)3S)(CO)6. The dramatically increased rate of electrocatalysis obtained from 4Fe6S over all previously identified model compounds appears to be related to the features uniquely common between it and the H-cluster, namely, that turnover involves the same formal redox states of the diiron unit (FeIFeII and Fe0FeII), the presence of an open site on the outer Fe atom of the Fe0FeII unit, and the thiolate-bridge to a second one-electron redox unit.     

QM/MM-PBSA method to estimate free energies for reactions in proteins

We have developed a method to estimate free energies of reactions in proteins, called QM/MM-PBSA. It estimates the internal energy of the reactive site by quantum mechanical (QM) calculations, whereas bonded, electrostatic, and van der Waals interactions with the surrounding protein are calculated at the molecular mechanics (MM) level. The electrostatic part of the solvation energy of the reactant and the product is estimated by solving the Poisson-Boltzmann (PB) equation, and the nonpolar part of the solvation energy is estimated from the change in solvent-accessible surface area (SA). Finally, the change in entropy is estimated from the vibrational frequencies. We test this method for five proton-transfer reactions in the active sites of [Ni,Fe] hydrogenase and copper nitrite reductase. We show that QM/MM-PBSA reproduces the results of a strict QM/MM free-energy perturbation method with a mean absolute deviation (MAD) of 8-10 kJ/mol if snapshots from molecular dynamics simulations are used and 4-14 kJ/mol if a single QM/MM structure is used. This is appreciably better than the original QM/MM results or if the QM energies are supplemented with a point-charge model, a self-consistent reaction field, or a PB model of the protein and the solvent, which give MADs of 22-36 kJ/mol for the same test set.     

Fe-S complexes containing five-membered heterocycles: novel models for the active site of hydrogenases with unusual low reduction potential

Three biomimetic 2Fe2S complexes [{(micro-SCH2)2NCH2(2-C4H3O)}](Fe2(CO)6), [{(micro-SCH2)2 NCH2(2-C4H3S)}](Fe2(CO)6) and [{(micro-SCH2)2NCH2(5-Br-2-C4H2S)}Fe2(CO)6] were prepared as models for the active site of Fe-only hydrogenase by the convergent process from [(micro-S2)Fe2(CO)6] and N,N-bis(hydromethyl)-2-furan and thiophene. The structures of these complexes were identified spectroscopically and crystallographically. The electrochemical behavior of the complexes and was unique as they showed catalytic proton reduction with a low reduction potential at -1.13 and -1.09 V vs Fc/Fc+, respectively, in the presence of HClO4.     

Carbon Monoxide and Cyanide Ligands in a Classical Organometallic Complex Model for Fe-Only Hydrogenase

The Fe(I) organometallic complex [(μ-SCH(2)CH(2)CH(2)S)Fe(2)(CO)(6)] provides a structural model for the cyano-carbonyl diiron site of Fe-only hydrogenase as characterized by X-ray crystallography (the picture shows the structure (black) of the model overlaid with that of the Fe-Fe dimetallic site in the hydrogenase isolated from Desulfovibrio desulfuricans). Cyanide substitution of CO occurs readily and provides spectroscopic references for the active site.     

Theoretical study of N2O reduction by CO in Fe-BEA Zeolite.

Quantum mechanical (QM) and QM/molecular mechanics (MM) studies of the full catalytic cycle of N(2)O reduction by CO in Fe-BEA zeolite, that is, oxidation of BEA-Fe by N(2)O and reduction of BEA-Fe-alphaO by CO, is presented. A large QM cluster, representing half of the channel of the BEA zeolite, is used. The contribution of the MM embedding to the calculated activation energies is found to be negligible. The minimum-energy paths for N(2)O decomposition and reduction with CO are calculated using the nudged elastic band (NEB) method. Calculated and experimental activation energies are in good agreement. The two possible orientations for the gaseous molecules adsorbing on the Fe site that are found lead to different activation energies.     

Artificial hydrogenases: assembly of an H-cluster analogue within a functionalised poly(pyrrole) matrix

We show that a redox active {Fe(4)S(4)}(2+)-cubane assembly covalently bound within a cysteinyl-alkylammonium functionalised polypyrrole can be modified with a diiron dithiolate carbonyl unit to give an artificial hydrogenase H-cluster framework confined within the polymer matrix.     

Density functional study on dihydrogen activation at the H cluster in Fe-only hydrogenases

Models simulating the catalytic diiron subcluster [FeFe](H) in Fe-only hydrogenases have often been designed for computational exploration of the catalytic mechanism of the formation and cleavage of dihydrogen. In this work, we extended the above models by explicitly considering the electron reservoir [4Fe-4S](H) which is linked to the diiron subcluster to form a whole H cluster ([6Fe-6S] = [4Fe-4S](H) + [FeFe](H)). Large-scale density functional theory (DFT) computations on the complete H cluster, together with simplified models in which the [4Fe-4S](H) subcluster is not directly involved in the reaction processes, have been performed to probe hydrogen activation on the Fe-only hydrogenases. A new intermediate state containing an Fe(p)...H...CN two-electron three-center bond is identified as a key player in the H2 formation/cleavage processes.     

Computational chemical analysis of [FeFe] hydrogenase H-cluster analogues to discern catalytically relevant features of the natural diatomic ligand configuration

Density functional theoretical models of the electronic structure of several configurational isomers and analogues of the [2Fe](H) H-cluster in [FeFe] hydrogenase were analyzed to identify distinguishing features of the canonical cofactor structure potentially relevant to catalysis. Collective analysis of geometric changes over models of oxidized and reduced [2Fe] clusters highlighted movement of the bridging carbonyl and anticorrelation of the proximal and distal Fe-C(terminal) bonds as key explanatory factors for variance over the considered models. Charge and bond order analysis suggest that as the bridging carbonyl favors the distal iron upon reduction, bonding simultaneously becomes more ionic in nature, raising the possibility of simple electrostatic stabilization as a factor in charge accumulation prior to ultimate H(2) creation and release. Frontier orbital energies show cis and trans arrangements of cyanide on the Fe-Fe core to have distinctive energies from the other models, which may be important for redox poise. Altogether, few factors qualitatively distinguish the cis- from the trans-cyano configurations, which may in fact enhance catalytic robustness under conditions leading to exchange of the bridging and terminal carbonyl ligands. However, the naturally occurring trans configuration possesses two distinct donor-metal-acceptor S-Fe-C(O) interactions, which might play a role in enforcing a low-spin ground state for the hydridic mechanism of H(2) production.     

In Vivo EPR Characterization of Semi‐Synthetic [FeFe] Hydrogenases

EPR spectroscopy reveals the formation of two different semi‐synthetic hydrogenases in vivo. [FeFe] hydrogenases are metalloenzymes that catalyze the interconversion of molecular hydrogen and protons. The reaction is catalyzed by the H‐cluster, consisting of a canonical iron–sulfur cluster and an organometallic [2Fe] subsite. It was recently shown that the enzyme can be reconstituted with synthetic cofactors mimicking the composition of the [2Fe] subsite, resulting in semi‐synthetic hydrogenases. Herein, we employ EPR spectroscopy to monitor the formation of two such semi‐synthetic enzymes in whole cells. The study provides the first spectroscopic characterization of semi‐synthetic hydrogenases in vivo, and the observation of two different oxidized states of the H‐cluster under intracellular conditions. Moreover, these findings underscore how synthetic chemistry can be a powerful tool for manipulation and examination of the hydrogenase enzyme under in vivo conditions.