A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation†

Abstract

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe₇S₉MoC-homocitrate) as a critical N₂ binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E₄(4H), which has accumulated 4[e^?/H⁺] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E₄(4H) is poised to bind/reduce N₂ as driven by mechanistically-coupled H₂ reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H₂ as the enzyme relaxes to state E₂(2H), containing 2[e^?/H⁺] as a hydride and sulfur-bound proton; accumulation of E₄(4H) in α-V70I is enhanced by HP suppression. EPR and ⁹⁵Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e^?/H⁺] to the E₀ state of the WT MoFe protein and to both α-V70I conformations generating E₂(2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e^?/H⁺] generates E₄(4H) with Fe2–H–Fe6 as the second hydride. E₄(4H) in WT enzyme and a minority α-V70I E₄(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E₂(2H) containing Fe3–H–Fe7. In the dominant α-V70I E₄(4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E₂(2H) contains Fe2–H–Fe6. It is this HP suppression in E₄(4H) that enables α-V70I MoFe to accumulate E₄(4H) in high occupancy. In addition, HP suppression in α-V70I E₄(4H) kinetically unmasks hydride reductive-elimination without N₂-binding, a process that is precluded in WT enzyme.