A model for dinitrogen binding in the E4 state of nitrogenase

Abstract

Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic iron–molybdenum–sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N₂-binding E₄ state is not known. In this study we present theoretical QM/MM calculations of new structural models of the E₄ 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 E₄ state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe₂ and Fe₆ with a terminal sulfhydryl group on either Fe₂ or Fe₆ (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe₂ and Fe₆. 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 E₄ 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 Fe₂ or Fe₆ 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 N₂ 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 Fe₂ or Fe₆ that partially activates N₂. We speculate that stabilization of an N₂-bound Fe(I) at Fe₆ (not found for Fe₂ model) via reductive elimination is a crucial part of N₂ 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 Fe₂ and Fe₆) 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 Fe₆-bound model than the Fe₂-bound model; however, this protonation is uphill in energy, suggesting protonation of N₂ might occur later in the catalytic cycle or via another mechanism.