Understanding non-reducible N in the mechanism of Mo-nitrogenase.

In my proposed mechanism of Mo-nitrogenase there are two roles for separate N molecules. One N diffuses into the reaction zone between Fe2 and Fe6 where a strategic gallery of H atoms can capture N to form the Fe-bound HNNH intermediate which is then progressively hydrogenated through intermediates containing HNNH, NH and NH entities and then two NH in sequence. The second N can be parked in an N-pocket about 3.

The mechanism of Mo-nitrogenase: from N capture to first release of NH.

Mo-nitrogenase hydrogenates N to NH. This report continues from the previous paper [I. Dance, , 2024, , 14193-14211] that described how the active site FeMo-co of the enzyme is uniquely able to capture and activate N, forming a key intermediate with Fe-bound HNNH.

The activating capture of N at the active site of Mo-nitrogenase.

Dinitrogen is inherently inert. This report describes detailed density functional calculations (with a 485+ atom model) of mechanistic steps by which the enzyme nitrogenase activates unreactive N at the intact active site FeMo-co, to form a key intermediate with bound HNNH. This mechanism does not bind N first and then add H atoms, but rather captures N ('N2-ready') that diffuses in through the substrate channel and enters a strategic gallery of H atom donors in the reaction zone, between Fe2 and Fe6.

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

In converting N to NH the enzyme nitrogenase utilises 8 electrons and 8 protons in the complete catalytic cycle. The source of the electrons is an FeS 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.

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

The binding of N to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH, but also has a separate role in promoting the N-dependent HD reaction (D + 2H + 2e → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N, directly towards the -coordination position of Fe2, a position which is outside the catalytic reaction domain. This led to the hypothesis [I.

The HD Reaction of Nitrogenase: a Detailed Mechanism.

Nitrogenase is the enzyme that converts N to NH under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo-co [Fe S CMo(homocitrate)] is unknown. An obligatory co-product is H , while exogenous H is a competitive inhibitor.

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 FeMoSC 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.

Calculating the chemical mechanism of nitrogenase: new working hypotheses.

The enzyme nitrogenase converts N to NH with stoichiometry N + 8H + 8e → 2NH + H. The mechanism is chemically complex with multiple steps that must be consistent with much accumulated experimental information, including exchange of H and N and the N-dependent hydrogenation of D to HD. Previous investigations have developed a collection of working hypotheses that guide ongoing density functional investigations of mechanistic steps and sequences.

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

The chemical reactions occurring at the FeMoSC(homocitrate) cluster, FeMo-co, the active site of the enzyme nitrogenase (N → NH), are enigmatic. Experimental information collected over a long period reveals aspects of the roles of N and H, each with more than one type of reactivity. This paper reports investigations of the binding of H and N at intact FeMo-co, using density functional simulations of a large 486 atom relevant portion of the protein, resulting in 27 new structures containing H and/or N bound at the and coordination sites of the participating Fe atoms, Fe2 and Fe6.

Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase.

The chemical mechanism of nitrogenase, catalysing N +8 e+8 H →2 NH +H , occurs at a large multi-metal cluster (FeMo-co) with composition CFe MoS (homocitrate). More than 20 steps are required. Experimental elucidation of this mechanism is elusive, for various reasons, and computational approaches have a valuable role.

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 (N→ NH) is a FeMoSC 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.

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

The enzyme nitrogenase converts N to NH , 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 NH . A separate small water pool (SWP), in a different part of the protein surrounding FeMo-co, has unknown function.

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

The active site of the enzyme nitrogenase is the FeMo-cofactor (FeMo-co), a C-centred FeMoS cluster, connected to the surrounding MoFe protein via ligands Cys and His. Density functional calculations, involving 14 additional surrounding amino acids, focus on His because its mutation causes important reactivity changes, including almost complete loss of ability to reduce N to NH. The Nε side-chain of His is capable of hydrogen bonding to S2B, bridging Fe2 and Fe6 of FeMo-co, believed to be the main reaction domain of nitrogenase.

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.

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.

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.

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.

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, H → 2H + 2e, that occurs at the Ni-Fe catalytic site. This site is normally inactivated by O, but the subclass of O-tolerant [NiFe] hydrogenases are able to counter this inactivation through the agency of an unusual and unprecedented proximal cluster, with composition [FeS(S)], that is able to transfer two electrons back to the Ni-Fe site and effect crucial reduction of O-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 O attack.

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.

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.

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.

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 controlled relay of multiple protons required at the active site of nitrogenase.

The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described.

Ramifications of C-centering rather than N-centering of the active site FeMo-co of the enzyme nitrogenase.

Nitrogenase catalyses the hydrogenation of N(2) to NH(3), and of CO to hydrocarbons. The active site is FeMo-co, an Fe(7)MoS(9) cluster with an atom (X(c)) at the centre of the inner trigonal prism of six Fe atoms. Calculations extending over almost a decade yielded consensus that this atom was nitrogen.