Electron Redistribution within the Nitrogenase Active Site FeMo-Cofactor During Reductive Elimination of H₂ to...

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E₄(4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E₄(4H) hydrides showed that enzymatic re of E₄(4H) hydride yields an H₂-bound complex (E₄(H₂,2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe-H bonds to the metal ions themselves enables N₂ to bind with concomitant H₂ release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E₄(H₂,2H) has not been trapped, cryogenic photolysis of E₄(4H) provides a means to address this question. Photolysis of E₄(4H) causes hydride-re with release of H₂, generating doubly reduced FeMo-co (denoted E₄(2H)*), the extreme limit of the electron-density redistribution upon formation of E₄(H₂,2H). Here we examine the doubly reduced FeMo-co core of the E₄(2H)* limiting-state by ¹H, ⁵⁷Fe, and ⁹⁵Mo ENDOR to illuminate the partial electron-density redistribution upon E₄(H₂,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E₄(2H)* ENDOR results as extended by DFT computations include (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; and (ii) two distinctive E₄(4H) ⁵⁷Fe signals are suggested as associated with structurally identified "anchors" of one bridging hydride, two others with identified anchors of the second, with NBO-analysis further identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe-H bonds.