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

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-C-homocitrate] 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/oxidative-addition 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.