PNNL

Abstract

Study of a-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding 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 two 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 a-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state a-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 are visualized as reflecting two conformations of the Ile residue, as seen in a reanalysis of the X-ray diffraction data of a-V70I. EPR measurements show delivery of 2[e-/H+] to WT MoFe protein and to both a-V70I conformations generates E2(2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e-/H+] generates the second hydride of E4(4H), Fe2-H-Fe6. E4(4H) in WT enzyme and presumably the unperturbed a-V70I conformation relaxes to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by HP of Fe3-H-Fe7, which leads to transient accumulation of E2(2H) containing Fe3-H-Fe7. In the second, perturbed a-V70I conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; HP of Fe3-H-Fe7 occurs first and the resulting E2(2H) contains Fe2-H-Fe6. It is this HP suppression that enables a-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in a-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.