Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N ₂ reduction

Abstract

Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N 2 → 2NH 3 reduction involves the reductive elimination (re) of H 2 from two [Fe-H-Fe] bridging hydrides bound to the catalytic [7Fe-9S-Mo-Chomocitrate] FeMo-cofactor (FeMo-co). These studies rationalize the Lowe-Thorneley kinetic scheme's proposal of mechanistically obligatory formation of one H 2 for each N 2 reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i) This shows that to prevent spurious disruption of FeMo-co having accumulated 4[e − /H + ] it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii) These calculations indicate an important role of sulfide hemilability in the overall conversion of E 0 to a diazene-level intermediate. (iii ) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H 2 formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutral re/oxidativeaddition equilibrium, (iiib) while preventing the "futile" generation of two H 2 without N 2 reduction: hydride re generates an H 2 complex, but H 2 is only lost when displaced by N 2 , to form an end-on N 2 complex that proceeds to a diazene-level intermediate.iological nitrogen fixation is one of the most challenging chemical transformations in biology, the conversion of N 2 to ammonia. The catalyst for biological N 2 fixation, the metalloenzyme nitrogenase, is known in three different forms (1) with the most abundant being the Mo-dependent enzyme, which is composed of the electron-donating Fe protein and the catalytic MoFe protein. The Fe protein, a homodimer, delivers one electron at a time during transient association with the MoFe protein heterotetramer with dissociation of the two proteins driven by the hydrolysis of two ATP to two ADP/P i per electron transfer (ET) event (2). The reduction of N 2 takes place on the [7Fe-9S-Mo-C-homocitrate] FeMo-cofactor (FeMo-co) in the active site of the MoFe protein (Fig. 1), with the [8Fe-7S] Pcluster in the MoFe protein acting as an ET intermediary.Lowe and Thorneley put forward a kinetic model for catalysis by the MoFe protein, including rate constants for formation of each intermediate state, designated as E n , where n indicates the number of electrons and protons accumulated. This scheme incorporates a controversial limiting stoichiometry:with an obligatory formation of 1 mol of H 2 per mole of N 2 reduced, and a corresponding requirement of 8[e − /H + ], ...

A model for dinitrogen binding in the E4state of nitrogenase

“…This energetic analysis suggests that models j-o are the most viable models for the E 4 state (at the TPSSh-QM/MM level). Open sulde-bridge models like j-o were not considered in the study by Cao et al 27 but some open-sulde bridge models were discussed by Raugei et al 24 and were found to be similar in energy as model e, which, however, is not in agreement with our results. We attribute this disagreement to different modelling aspects, i.e.…”

“…All models that we consider in this study were structurally optimized at the same QM/MM level of theory that we have previously used to describe the resting state E 0 and we considered multiple broken-symmetry states for each model. As recently discussed by Raugei et al, 24 the protein environment and the quality of the computational model can make a surprisingly large difference regarding the relative stability of an E 4 isomer. Raugei et al demonstrated e.g.…”

“…Mutation studies have previously implicated the Fe 2 -Fe 3 -Fe 6 -Fe 7 face as a likely binding site for dinitrogen as no dinitrogen reduction is observed when the residue a-70 Val is mutated into the bulkier a-70 Ile . 22 Recent joint experimentalcomputational studies have proposed E 4 models featuring bridging hydrides [23][24][25] (the favoured model features bridging hydrides between Fe 3 -Fe 7 and Fe 2 -Fe 6 ) while other computational studies have suggested mixed bridging/terminal hydride models 26,27 or even, surprisingly, structures featuring a protonated carbide with/without bridging hydrides. [28][29][30][31] Additionally, recent crystal structures of a CO-bound form 32 of MoFe protein (CO being an inhibitor) and an XH-ligand-bound (X ¼ N or O) form 33 of VFe protein have emerged, that show CO/XH replacing the Fe 2 -Fe 6 bridging sulde in a bridging binding mode (we recently showed via QM/MM calculations that the XH ligand in the FeVco structure is more consistent with an OH).…”

“…As some of the models in this study feature structural changes at these Fe ions, it seems possible that such a solution could become more favourable than other BS solutions. To aid in the computational problem involving many models and many BS states at an expensive level of theory, we have restricted ourselves to performing geometry optimizations with the BS-235, BS-247, BS-346, BS-147 solutions to explore the energetics of all models in Fig Models n, m and o were previously discussed as models close in energy to the favoured model e in work by Raugei et al In this study we introduce models l and model o (though a model like o has been discussed by Raugei et al 24 ) as plausible candidates for E 4 as well as models j and k, being similar to models m and n, and model b featuring two terminal sulydryl groups and two bridging hydrides. Model i also has a terminal sulydryl group like l but has a different sulde protonation state.…”

Predicting Ligand-Dissociation Energies of 3d Coordination Complexes with Auxiliary-Field Quantum Monte Carlo

“…in clusters of Mn atoms in Photosystem II (PSII) or Fe and Mo atoms in nitrogenases. [4][5][6][7] Furthermore, the coordination of small molecules to single metal ions is an important motif in drug design, 8 and the correlations exhibited in the copper oxide layers of cuprate materials play a central role in the phenomenon of high-temperature superconductivity. 9,10 Ab initio modeling has the potential to yield essential insights into these transition metal systems.…”