Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic ironmolybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N 2 -binding E 4 state is not known. In this study we present theoretical QM/ MM calculations of new structural models of the E 4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E 4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe 2 and Fe 6 with a terminal sulfhydryl group on either Fe 2 or Fe 6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe 2 and Fe 6 . These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E 4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly different proposals for this state. Importantly, dinitrogen binds exothermically to either Fe 2 or Fe 6 in our models, contrary to others, an effect rationalized via the unique ligand field (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N 2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe 2 or Fe 6 that partially activates N 2 . We speculate that stabilization of an N 2 -bound Fe(I) at Fe 6 (not found for Fe 2 model) via reductive elimination is a crucial part of N 2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the sulfhydryl group (to regenerate the sulfide bridge between Fe 2 and Fe 6 ) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more favorable with the Fe 6 -bound model than the Fe 2 -bound model; however, this protonation is uphill in energy, suggesting protonation of N 2 might occur later in the catalytic cycle or via another mechanism. Electronic supplementary information (ESI) available: Further information on all broken-symmetry solutions calculated for all models. Details of the QM/MM model preparation. Spin populations of all models. Localized orbital analysis of selected models. Geometry analysis of E 0 state calculated with different functionals and electronic structure analysis. Cartesian coordinates for the QM regions of all optimized structures available as XYZ les. See

The FeMoco cluster of Mo nitrogenase undergoes minor distortions upon reduction to E1, supporting iron-based reduction and belt sulfide protonation.

Molybdenum-dependent nitrogenase is the most active biological catalyst for dinitrogen reduction. This reaction is catalyzed by a [MoFe 7 S 9 C] cofactor (FeMoco). FeMoco can be described as a double-cubane, with [MoFe 3 S 3 ] and [Fe 4 S 3 ] parts, bound via an interstitial carbide and three bridging sulfides. Model compounds have been synthesized since early studies of the enzyme and Coucouvanis and co-workers demonstrated that [MoFe 3 S 4 ] cubanes are active catalysts for many substrates catalyzed by nitrogenase. These reactions include hydrazine reduction to ammonia and cis-dimethyldiazene reduction to methylamine. Experiments implicated molybdenum as the binding site but the mechanisms have not been studied by theoretical calculations before. Here we present a DFT study of the catalytic reaction mechanisms of hydrazine and cis-dimethyldiazene reduction with a [MoFe 3 S 4 ] cubane. Like in the experiments, molybdenum is revealed as the likely substrate binding site, likely due to the labile ligand on Mo. For the hydrazine mechanism, a reduction event is centered on Fe, specifically on the Fe antiferromagnetically coupled to the mixed-valence pair. After protonation of the distal hydrazine nitrogen, the N−N bond can be cleaved to yield NH 3 and a Mo-bound -NH 2 intermediate. This is followed by another protonation/reduction step to give an -NH 3 intermediate, and finally substituted by the substrate to complete the cycle. The computed mechanisms shed light on the bimetallic cooperativity in these systems where the reduction steps are localized on Fe while the substrate binds to Mo and the reductions require only a free coordination site (on Mo) and a favorable reduction event (to Fe). Although both substrates easily displace the weakly bound acetonitrile ligand, one reduction event is required for hydrazine activation and N−N bond cleavage to give an integer-spin -NH 2 intermediate. An integer-spin -NH 2 intermediate has been observed as a common intermediate for the enzyme reduction of hydrazine and diazene, suggesting a possible link to the enzyme chemistry.