What triggers the coupling of proton transfer and electron transfer at the active site of nitrogenase?

In converting N2 to NH3 the enzyme nitrogenase utilises 8 electrons and 8 protons in the complete catalytic cycle. The source of the electrons is an Fe4S4 reductase protein (Fe-protein) which temporarily docks with the MoFe-protein that contains the catalytic active cofactor, FeMo-co, and an electron transfer cluster called the P cluster. The overall mechanism involves 8 repetitions of a cycle in which reduced Fe-protein docks with the MoFe-protein, one electron transfers to the P-cluster, and then to FeMo-co, followed by dissociation of the two proteins and re-reduction of the Fe-protein. Protons are supplied serially to FeMo-co by a Grotthuss proton translocation mechanism from the protein surface along a conserved chain of water molecules (a proton wire) that terminates near S atoms of the FeMo-co cluster [CFe7S9Mo(homocitrate)] where the multiple steps of the chemical conversions are effected. It is assumed that the chemical mechanisms use proton-coupled electron-transfer (PCET) and that H atoms (e- + H+) are involved in each of the hydrogenation steps. However there is neither evidence for, or mechanism proposed, for this coupling. Here I report calculations of cluster charge distribution upon electron addition, revealing that the added negative charge is on the S atoms of FeMo-co, which thereby become more basic, and able to trigger proton transfer from H3O+ waiting at the near end of the proton wire. This mechanism is supported by calculations of the dynamics of the proton transfer step, in which the barrier is reduced by ca. 3.5 kcal mol-1 and the product stabilised by ca. 7 kcal mol-1 upon electron addition. H tunneling is probable in this step. In nitrogenase it is electron transfer that triggers proton transfer.

The binding of reducible N2 in the reaction domain of nitrogenase.

The binding of N2 to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH3, but also has a separate role in promoting the N2-dependent HD reaction (D2 + 2H+ + 2e- → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N2, directly towards the exo-coordination position of Fe2, a position which is outside the catalytic reaction domain. This led to the hypothesis [I. Dance, Dalton Trans., 2022, 51, 12717] of 'promotional' N2 bound at exo-Fe2, and a second 'reducible' N2 bound in the reaction domain, specifically the endo-coordination position of Fe2 or Fe6. The range of possibilities for the binding of reducible N2 in the presence of bound promotional N2 is described here, using density functional simulations with a 486 atom model of the active site and surrounding protein. The pathway for ingress of the second N2 through protein, past the first N2 at exo-Fe2, and tumbling into the binding domain between Fe2 and Fe6, is described. The calculations explore 24 structures involving 6 different forms of hydrogenated FeMo-co, including structures with S2BH unhooked from Fe2 but tethered to Fe6. The calculations use the most probable electronic states. End-on (η1) binding of N2 at the endo position of either Fe2 or Fe6 is almost invariably exothermic, with binding potential energies ranging up to -18 kcal mol-1. Many structures have binding energies in the range -6 to -14 kcal mol-1. The relevant entropic penalty for N2 binding from a diffusible position within the protein is estimated to be 4 kcal mol-1, and so the binding free energies for reducible N2 are suitably negative. N2 binding at endo-Fe2 is stronger than at endo-Fe6 in three of the six structure categories. In many cases the reaction domain containing reducible N2 is expanded. These results inform computational simulation of the subsequent steps in which surrounding H atoms transfer to reducible N2.

The HD Reaction of Nitrogenase: a Detailed Mechanism.

Nitrogenase is the enzyme that converts N2 to NH3 under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo-co [Fe7 S9 CMo(homocitrate)] is unknown. An obligatory co-product is H2 , while exogenous H2 is a competitive inhibitor. Isotopic substitution using exogenous D2 revealed the N2 -dependent reaction D2 +2H+ +2e- →2HD (the 'HD reaction'), together with a collection of additional experimental characteristics and requirements. This paper describes a detailed mechanism for the HD reaction, developed and elaborated using density functional simulations with a 486-atom model of the active site and surrounding protein. First D2 binds at one Fe atom (endo-Fe6 coordination position), where it is flanked by H-Fe6 (exo position) and H-Fe2 (endo position). Then there is synchronous transfer of these two H atoms to bound D2 , forming one HD bound to Fe2 and a second HD bound to Fe6. These two HD dissociate sequentially. The final phase is recovery of the two flanking H atoms. These H atoms are generated, sequentially, by translocation of a proton from the protein surface to S3B of FeMo-co and combination with introduced electrons. The first H atom migrates from S3B to exo-Fe6 and the second from S3B to endo-Fe2. Reaction energies and kinetic barriers are reported for all steps. This mechanism accounts for the experimental data: (a) stoichiometry; (b) the N2 -dependence results from promotional N2 bound at exo-Fe2; (c) different N2 binding Km for the HD reaction and the NH3 formation reaction results from involvement of two different sites; (d) inhibition by CO; (e) the non-occurrence of 2HD→H2 +D2 results from the synchronicity of the two transfers of H to D2 ; (f) inhibition of HD production at high pN2 is by competitive binding of N2 at endo-Fe6; (g) the non-leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction.

Understanding the tethered unhooking and rehooking of S2B in the reaction domain of FeMo-co, the active site of nitrogenase.

The active site of the nitrogen fixing enzyme nitrogenase is an Fe7MoS9C cluster, and investigations of the enigmatic chemical mechanism of the enzyme have focussed on a pair of Fe atoms, Fe2 and Fe6, and the S2B atom that bridges them. There are three proposals for the status of the Fe2-S2B-Fe6 bridge during the catalytic cycle: one that it remains intact, another that it is completely labile and absent during catalysis, and a third that S2B is hemilabile, unhooking one of its bonds to Fe2 or Fe6. This report examines the tethered unhooking of S2B and factors that affect it, using DFT calculations of 50 geometric/electronic possibilities with a 485 atom model including all relevant parts of surrounding protein. The outcomes are: (a) unhooking the S2B-Fe2 bond is feasible and favourable, but alternative unhooking of the S2B-Fe6 bond is unlikely for steric reasons, (b) energy differences between hooked and unhooked isomers are generally <10 kcal mol-1, usually with unhooked more stable, (c) ligation at the exo-Fe6 position inhibits unhooking, (d) unhooking of hydrogenated S2B is more favourable than that of bare S2B, (e) hydrogen bonding from the NεH function of His195 to S2B occurs in hooked and unhooked forms, and possibly stabilises unhooking, (f) unhooking is reversible with kinetic barriers ranging 10-13 kcal mol-1. The conclusion is that energetically accessible reversible unhooking of S2B or S2BH, as an intrinsic property of FeMo-co, needs to be considered in the formulation of mechanisms for the reactions of nitrogenase.

Calculating the chemical mechanism of nitrogenase: new working hypotheses.

The enzyme nitrogenase converts N2 to NH3 with stoichiometry N2 + 8H+ + 8e- → 2NH3 + H2. The mechanism is chemically complex with multiple steps that must be consistent with much accumulated experimental information, including exchange of H2 and N2 and the N2-dependent hydrogenation of D2 to HD. Previous investigations have developed a collection of working hypotheses that guide ongoing density functional investigations of mechanistic steps and sequences. These include (i) hypotheses about the serial provision of protons and their conversion to H atoms bonded to S and Fe atoms of the FeMo-co catalytic site, (ii) the migration of H atoms over the surface of FeMo-co, (iii) the roles of His195, (iv) identification of three protein channels, one for the ingress of N2, a separate pathway for the passage of exogenous H2 (D2) and product H2 (HD), and a hydrophilic pathway for egress of product NH3. Two additional working hypotheses are described in this paper. N2 passing along the N2 channel approaches and binds end-on to the exo coordination position of Fe2, with favourable energetics when FeMo-co is pre-hydrogenated. This exo-Fe2-N2 is apparently not reduced but has a promotional role by expanding the reaction zone. A second N2 can enter via the N2 ingress channel and bind at the endo-Fe6 position, where it is surrounded by H atom donors suitable for the N2 → NH3 conversion. It is proposed that this endo-Fe6 position is also the binding site for H2 (generated or exogenous), accounting for the competitive inhibition of N2 reduction by H2. The HD reaction occurs at the endo-Fe6 site, promoted by N2 at the exo-Fe2 site. The second hypothesis concerns the most stable electronic states of FeMo-co with ligands bound at Fe2 and Fe6, and provides a protocol for management of electronic states in mechanism calculations.

Structures and reaction dynamics of N2 and H2 binding at FeMo-co, the active site of nitrogenase.

The chemical reactions occurring at the Fe7MoS9C(homocitrate) cluster, FeMo-co, the active site of the enzyme nitrogenase (N2 → NH3), are enigmatic. Experimental information collected over a long period reveals aspects of the roles of N2 and H2, each with more than one type of reactivity. This paper reports investigations of the binding of H2 and N2 at intact FeMo-co, using density functional simulations of a large 486 atom relevant portion of the protein, resulting in 27 new structures containing H2 and/or N2 bound at the exo and endo coordination sites of the participating Fe atoms, Fe2 and Fe6. Binding energies and transition states for association/dissociation are determined, and trajectories for the approach, binding and separation of H2/N2 are described, including diffusion of these small molecules through proximal protein. Influences of surrounding amino acids are identified. FeMo-co deforms geometrically when binding H2 or N2, and a procedure for calculating the energy cost involved, the adaptation energy, is introduced here. Adaptation energies, which range from 7 to 36 kcal mol-1 for the reported structures, are influenced by the protonation state of the His195 side chain. Seven N2 structures and three H2 structures have negative binding free energies, which include the estimated entropy penalties for binding of N2, H2 from proximal protein. These favoured structures have N2 bound end-on at exo-Fe2, exo-Fe6 and endo-Fe2 positions of FeMo-co, and H2 bound at the endo-Fe2 position. Various postulated structures with N2 bridging Fe2 and Fe6 revert to end-on-N2 at endo positions. The structures are also assessed via the calculated potential energy barriers for association and dissociation. Barriers to the binding of H2 range from 1 to 20 kcal mol-1 and barriers to dissociation of H2 range from 3 to 18 kcal mol-1. Barriers to the binding of N2, in either side-on or end-on mode, range from 2 to 18 kcal mol-1, while dissociation of bound N2 encounters barriers of 3 to 8 kcal mol-1 for side-on bonding and 7 to 18 kcal mol-1 for end-on bonding. These results allow formulation of mechanisms for the H2/N2 exchange reaction, and three feasible mechanisms for associative exchange and three for dissociative exchange are identified. Consistent electronic structures and potential energy surfaces are maintained throughout. Changes in the spin populations of Fe2 and Fe6 connected with cluster deformation and with metal-ligand bond formation are identified.

Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase.

The chemical mechanism of nitrogenase, catalysing N2 +8 e+8 H+ →2 NH3 +H2 , occurs at a large multi-metal cluster (FeMo-co) with composition CFe7 MoS9 (homocitrate). More than 20 steps are required. Experimental elucidation of this mechanism is elusive, for various reasons, and computational approaches have a valuable role. This review critically surveys recent density functional calculations of the coordination chemistry and relevant reactions of FeMo-co within the protein surrounds. Topics covered include the accuracies and validation of the density functionals, the treatment of electronic structure, and the chemical models used. The components of mechanism are described, including the input of N2 , proton supply, the egress of NH3 , and the roles of surrounding protein. This leads to descriptions and evaluations of the overall mechanistic cycles proposed on the basis of density functional calculations. Finally, I discuss some current issues, and consider the outlook for further work.

How feasible is the reversible S-dissociation mechanism for the activation of FeMo-co, the catalytic site of nitrogenase?

The active site of the enzyme nitrogenase (N2→ NH3) is a Fe7MoS9C cluster that contains three doubly-bridging µ-S atoms around a central belt. A vanadium nitrogenase variant has a slightly different cluster, containing two µ-S atoms. Recent crystal structures have revealed substitution of one µ-S (S2B, bridging Fe2 and Fe6), by CO in Mo-nitrogenase and an uncertain light atom in V-nitrogenase. These systems retained catalytic activity, and were able to recover the lost µ-S atom. Electron density attributed to the dissociated S is displaced by 7 Å in the crystal structure of the non-standard V-protein. The hypothesis arising from these observations is that the chemical mechanism of nitrogenase involves reversible dissociation of S2B, leaving Fe2 and Fe6 seriously under-coordinated and reactive in trapping N2 and binding reaction intermediates. Accumulated experimental evidence points to the Fe2-S2B-Fe6 domain as the centre of catalytic hydrogenation of N2. Using DFT simulations of a large model (>488 atoms) containing all relevant surrounding protein residues, I have investigated the chemical steps that could allow dissociation of S2B. The participation of H atoms is crucial, as is involvement of the nearby side chain of His195 that can function as proton donor to S2B and hydrogen-bonding supporter of displaced S2B. A significant result is that after ingress and binding of N2 at Fe2 the breaking of the Fe2-S2B bond can be strongly exergonic with negligible kinetic barrier. Subsequent extension of the Fe6-S2B bond and dissociation as H2S (or SH-) is endergonic by 20-25 kcal mol-1, partly because the separating H2S is restricted by surrounding amino-acids. I present a number of reaction sequences and energy landscapes, and derive thirteen chemical principles relevant to the postulated S-dissociation mechanism. A key conclusion is that unhooking of S2BH or S2BH2 from Fe2 is favourable, likely, and propitious for subsequent H transfer to bound N2 or reaction intermediates. The space between Fe2 and Fe6 supports two bridging ligands, and another H atom on Fe6 can move without kinetic barrier to occupy the bridging position vacated by S2B.

What is the role of the isolated small water pool near FeMo-co, the active site of nitrogenase?

The enzyme nitrogenase converts N2 to NH3 , and hydrogenates many other small unsaturated molecules, using multiple electrons and multiple protons. The protein contains a number of water structures in the vicinity of the active site, FeMo-co, and functional roles have been assigned to two of these with detailed mechanisms proposed for the serial ingress of protons and the egress of product NH3 . A separate small water pool (SWP), in a different part of the protein surrounding FeMo-co, has unknown function. A recent investigation of protein crystals soaked in low-pH buffer revealed changes in residues near this SWP, and suggested that it could be involved in proton transfer steps. This paper examines the SWP in three protein crystal structures, Azotobacter vinelandii (Av1) and Clostridium pasterianum (Cp1) in their neutral resting states, and Cp1 at low pH. The H atoms, not observed crystallographically, were patched in through density functional calculations using large protein models. Optimisation of the various possibilities, with assessment against crystal dimensions, yielded the most probable distributions of hydrogen atoms in the hydrogen bonds, and the location of H3 O+ in the low-pH state. These detailed structures vary in water content and water involvement with surrounding residues, and vary also in their hydrogen bonding to S atoms of FeMo-co. A conserved mechanism for proton transfer to FeMo-co is not evident, and it is concluded that the SWP has no role in the mechanism of nitrogenase.

New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase.

The active site of the enzyme nitrogenase is the FeMo-cofactor (FeMo-co), a C-centred Fe7MoS9 cluster, connected to the surrounding MoFe protein via ligands Cys275 and His442. Density functional calculations, involving 14 additional surrounding amino acids, focus on His195 because its mutation causes important reactivity changes, including almost complete loss of ability to reduce N2 to NH3. The Nε side-chain of His195 is capable of hydrogen bonding to S2B, bridging Fe2 and Fe6 of FeMo-co, believed to be the main reaction domain of nitrogenase. Details are presented for the possible ways in which protonated or deprotonated Nε of His195 interact with S2B or S2B-H or Fe2 or Fe2-H or Fe-(H2). Movements of the His195 side-chain allow formation of a significant short dihydrogen bond between Nε of His195 and H on Fe2: Nε-H••H-Fe2, with H-H=1.39Å. It is shown that a 180° rotation of the imidazole ring of His195 is not able to facilitate transfer of protons from the protein surface to FeMo-co. His195 is able to move H atoms to and from S2B, and the characteristics of H transfer between S2B and Nε of His195 are described, together with their dependence on the protonation state of His195 and the redox state of FeMo-co. The water molecule on the posterior Nδ side of His195 can mediate proton transfer to and from the side-chain of Tyr228. The accumulated results suggest that protonated His195 could be the agent for the first, most difficult, transfer of H to bound substrate N2.

Mechanisms of the S/CO/Se interchange reactions at FeMo-co, the active site cluster of nitrogenase.

The active site of the N2 fixing enzyme nitrogenase is a C-centred Fe7MoS cluster (FeMo-co) containing a trigonal prism of six Fe atoms connected by a central belt of three doubly-bridging S atoms. The trigonal faces of the prism are capped via triply-bridging S atoms to Fe1 at one end and Mo at the other end. One of the central belt atoms, S2B, considered to be important in the chemical mechanism of the enzyme, has been shown by Spatzal, Rees et al. to undergo substitution by CO, and also substitution by Se in the presence of SeCN(-), under turnover conditions. Further, when turning over under C2H2 or N2/CO there is migration of Se to the other two belt bridging positions. These reactions are extraordinary, and unprecedented in metal chalcogenide cluster chemistry. Using density functional simulations, mechanisms for all of these reactions have been developed, involving the small molecules SCO, SeCO, C2H2S, C2H2Se, SeCN(-), SCN(-) functioning as carriers of S and Se atoms. The possibility that the S2B bridge position is vacant is discounted, because the barrier to formation of a bridge-void intermediate with two contiguous three-coordinate Fe atoms is too large. A bridging ligand is retained throughout the proposed mechanisms. Intermediates with Fe-C(O)-S/Se-Fe cycles and with SCO/SeCO C-bound to Fe are predicted. The energetics of the reaction trajectories show them to be feasible and easily reversible, consistent with experiment. Alternative mechanisms involving intramolecular differential rotatory rearrangements of the cluster to scramble the Se bridges are also examined, and shown to be very unlikely. The implications of these new facets of the reactivity of the FeMo-co cluster are discussed: it is considered that they are unlikely to be part of the mechanism of the physiological reactions of nitrogenase.

The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, <7 kcal mol(-1). During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The largest potential barrier is estimated to be 14 kcal mol(-1). These results are discussed in the context of reactivity data for nitrogenase, and the occurrence of a comparable water chain in cytochrome-c oxidase. Further investigation of the low-frequency conformational dynamics of the nitrogenase proteins, coupling proton transfer with other events in the nitrogenase cycle, is briefly canvassed.

Misconception of reductive elimination of H2, in the context of the mechanism of nitrogenase.

The elimination of H2 from an M(H)2 component of a coordination complex is often described as reductive elimination, in which the H atoms are regarded as hydride ions, and the product complex after elimination is regarded as reduced by two electrons. The concept is M(n+2)(H(-))2 → M(n) + H2 (with oxidative addition as its reverse). This interpretation contravenes Pauling's electroneutrality principle, and a number of researchers of metal-hydrogen systems have warned against literal acceptance of the formalism. A mechanism suggested by others for the chemical catalysis occurring at the Fe7MoS9C active site cluster of nitrogenase has invoked reductive elimination of H2 from Fe as a central premise. I report here calculations of atom partial charges for the relevant nitrogenase steps, as well as atom partial charges for some well-studied Fe complexes that model the nitrogenase chemistry. Fe-coordinated H atoms are <20% hydridic, and during the H2 elimination process the charge on Fe is essentially invariant. The argument for literal reductive elimination of H2 as part of the mechanism of nitrogenase is not sustained.

Protonation of bridging sulfur in cubanoid Fe4S4 clusters causes large geometric changes: the theory of geometric and electronic structure.

Density functional calculations indicate that protonation of a µ3-S atom in cubanoid clusters [Fe4S4X4](2-) leads to a large extension of one Fe-S(H) bond such that the SH ligand is doubly-bridging, µ-SH. Triply-bridging SH in these clusters is unstable, relative to µ-SH. The theory for the geometric and electronic structures of the protonated [Fe4S4X4](2-) clusters (X = Cl, SEt, SMe, SPh, OMe, OPh) is presented in this paper. The principal results are (1) the unique Fe atom in [Fe4S3(SH)X4](-) is three-coordinate, with planar or approximately planar stereochemistry, (2) approximately equi-energetic endo and exo isomers occur for pyramidal µ-SH, (3) the structural changes caused by protonation reverse without barrier on deprotonation, (4) the most stable electronic states have S = 0 and oppositely signed spin densities on the Fe atoms bearing the µ-SH bridge, (5) interconversions between endo and exo isomers, and between ground and excited states, occur through concerted lengthenings and shortenings of Fe-S(H) interactions, on relatively flat energy surfaces, (6) protonation of an X ligand does not change the characteristics of protonation of µ3-S. Experimental tests of this theory are suggested, and applications discussed.

What is the trigger mechanism for the reversal of electron flow in oxygen-tolerant [NiFe] hydrogenases?

The [NiFe] hydrogenases use an electron transfer relay of three FeS clusters - proximal, medial and distal - to release the electrons from the principal reaction, H2 → 2H+ + 2e-, that occurs at the Ni-Fe catalytic site. This site is normally inactivated by O2, but the subclass of O2-tolerant [NiFe] hydrogenases are able to counter this inactivation through the agency of an unusual and unprecedented proximal cluster, with composition [Fe4S3(Scys)6], that is able to transfer two electrons back to the Ni-Fe site and effect crucial reduction of O2-derived species and thereby reactivate the Ni-Fe site. This proximal cluster gates both the direction and the number of electrons flowing through it, and can reverse the normal flow during O2 attack. The unusual structures and redox potentials of the proximal cluster are known: a structural change in the proximal cluster causes changes in its electron-transfer potentials. Using protein structure analysis and density functional simulations, this paper identifies a closed protonic system comprising the proximal cluster, some contiguous residues, and a proton reservoir, and proposes that it is activated by O2-induced conformational change at the Ni-Fe site. This change is linked to a key histidine residue which then causes protonation of the proximal cluster, and migration of this proton to a key µ3-S atom. The resulting SH group causes the required structural change at the proximal cluster, modifying its redox potentials, and leads to the reversed electron flow back to the Ni-Fe site. This cycle is reversible, and the protons involved are independent of those used or produced in reactions at the active site. Existing experimental support for this model is cited, and new testing experiments are suggested.

Large structural changes upon protonation of Fe4S4 clusters: the consequences for reactivity.

Density functional calculations reveal that protonation of a µ3-S in [Fe4S4X4](2-) clusters (X = halide, thiolate, phenoxide) results in the breaking of one S-Fe bond (to >3 Å, from 2.3 Å). This creates a doubly-bridging SH ligand (µ3-SH is not stable), and a unique three-coordinated planar Fe atom. The under-coordination of this unique Fe atom is the basis of revised mechanisms for the acid-catalysed ligand substitution reactions in which substitution of X by PhS occurs at the unique Fe site by an indirect pathway involving initial displacement of X by acetonitrile (solvent), followed by displacement of coordinated acetonitrile by PhSH. When X = Cl or Br the rate of attack by PhSH is slower than the dissociation of X(-), and is the rate-determining step; in contrast, when X = SEt, SBu(t) or OPh the rate of dissociation of XH is slower than attack by PhSH and is rate-determining for these clusters. A full and consistent interpretation of all kinetic data is presented including new explanations of many of the kinetic observations on the acid-catalysed substitution reactions of [Fe4S4X4](2-) clusters. The proposed mechanisms are supported by density functional calculations of the structures of intermediates, and simulations of some of the steps. These findings are expected to have widespread ramifications for the reaction chemistry of both natural and synthetic clusters with the {Fe4S4} core.

Unexpected explanation for the enigmatic acid-catalysed reactivity of [Fe4S4X4](2-) clusters.

Density functional calculations show that Fe-S clusters undergo unexpected large structural changes when protonated at S. Protonation of prototypical cubanoid [Fe4S4X4](2-) to [Fe4S3(SH)X4](-) (X = Cl, SR, OR) results in formation of doubly-bridging SH, severance of one Fe-S bond, and creation of a three-coordinate Fe. These findings explain previously enigmatic results concerning the reactivity of these clusters, including the rates of protonation, pKa data, and the kinetics of acid-catalysed ligand substitution.

A molecular pathway for the egress of ammonia produced by nitrogenase.

Nitrogenase converts N2 to NH3, at one face of an Fe-Mo-S cluster (FeMo-co) buried in the protein. Through exploration of cavities in the structures of nitrogenase proteins, a pathway for the egress of ammonia from its generation site to the external medium is proposed. This pathway is conserved in the three species Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum. A molecular mechanism for the translocation of NH3 by skipping through a sequence of hydrogen bonds involving eleven water molecules and surrounding aminoacids has been developed. The putative mechanism requires movement aside of some water molecules by up to ~ 1Å. Consistent with this, the surrounding protein is comprised of different chains and has little secondary structure: protein fluctuations are part of the mechanism. This NH3 pathway is well separated from the water chain and embedded proton wire that have been proposed for serial supply of protons to FeMo-co. Verification procedures are suggested.

Nitrogenase: a general hydrogenator of small molecules.

Nitrogenase naturally converts N2 to NH3, but it also hydrogenates a variety of small molecules, in many cases requiring multiple electrons plus protons for each catalytic cycle. A general mechanism, arising from many density functional calculations and simulations, is proposed to account for all of these reactions. Protons, supplied serially in conjunction with electrons to the active site FeMo-co (a CFe7MoS9 (homocitrate) cluster), generate H atoms that migrate over and populate two S and two Fe atoms in the reaction domain. The mechanistic paradigm is conceptually straightforward: substrate (on Fe) and H atoms (on S and Fe) are bound contiguously in the reaction zone, and H atoms transfer (probably with some quantum tunneling) to the substrate to form product. Details and justifications of the mechanisms for N2 and other key substrates are summarised, and the unusual structure of FeMo-co as a general hydrogenation catalyst is rationalised. Testing experiments are suggested.

The stereochemistry and dynamics of the introduction of hydrogen atoms onto FeMo-co, the active site of nitrogenase.

The catalyzed hydrogenations effected at the active site FeMo-co of nitrogenase have been proposed to involve serial supply of the required multiple protons along a proton wire terminating at sulfur atom S3B of FeMo-co. In conjunction with serial electron transfer to FeMo-co, these protons become H atoms, and then are able to migrate from S3B to other Fe and S atoms of FeMo-co, and to transfer to bound substrate and intermediates. This general model, which can account for all reactions of nitrogenase, involves a preparatory stage in which each incoming H atom is required to move from the proton delivery side of S3B to the opposite migration side of S3B. This report examines the mechanism of this reconfiguration of S3B-H, finding four stable configurations in which S3B-H has pyramidal-trigonal coordination, with one elongated Fe-S3B interaction. The transition states and energies for reconfiguration are described. Pseudotetrahedral four coordination and planar-trigonal coordination for S3B-H are less stable than pyramidal-trigonal coordination. Results are presented for FeMo-co with one, two, three, and four H atoms (the E1H1, E2H2, E3H3, and E4H4 Thorneley-Lowe stages), and the general principles are defined, for application in the various chemical mechanisms of nitrogenase.