Photoinduced Reductive Elimination of H₂ from the Nitrogenase Dihydride (Janus) State Involves a FeMo-cofactor-H₂ Intermediate

Abstract

N2 reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe-H-Fe] bridging hydrides (denoted E4(4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the N≡N triple bond by the reductive elimination (re) of H2 from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that when E4(4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H2 to give a reactive doubly-reduced intermediate, denoted E4(2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H2 preceded N2 binding. Experiments reported here establish that photoinduced re occurs in two steps. Photolysis of E4(4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E4(2H)* + H2]. The experiments, supported by DFT calculation, indicate that the trapped intermediate is an H2 complex on the ground adiabatic potential energy suface that connects E4(4H) with [E4(2H)* + H2]. We suggest this complex, denoted E4(H2; 2H), is a thermally populated intermediate in the catalytically central re of H2 by E4(4H), and that N2 reacts with this complex to complete the activated conversion of [E4(4H) + N2] into [E4(2N2H) + H2].

Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E2(2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis

“…Our earlier conclusion that 1b is the E 2 (2H) state with its reducing equivalents stored as a bound hydride 4,6,9 is corroborated by the observation that the E 2 /1b state is photoactive, like inorganic hydride complexes, 17,18 the [Fe–H–Fe] bridging hydrides of the E 4 (4H) state of nitrogenase, 19,20 and the [Ni–H–Fe] bridging hydrides of the Ni–C state of NiFe hydrogenase. 21 As noted above, the earlier cryoannealing study of E 4 (4H) implied that the hydride of E 2 /1b adopts a [Fe–H–Fe] bridging structure, and it will be thus described in this report.…”

“…19,20 To complement this chemical difference, the latter process shows a much larger KIE ∼ 10, indicating the significance of tunneling in the H 2 formation.…”

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

“…10,11 The re mechanism is kinetically and thermodynamically reversible, 4,7 and the hydrides in the E 4 (4H) state are photolytically active. 5,6 In a parallel, competitive reaction, nitrogenase functions as a ‘hydrogenase’, producing H 2 through the protonation of a metal-hydride and relaxation to a two-electron less reduced E n-2 state (Figure 1B). …”

Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction

“…In recent years, we have demonstrated (6,7) that this stoichiometry arises because activation of nitrogenase for N 2 reduction involves the accumulation of four reducing equivalents at the active-site FeMo-co (4) to form a state with two [Fe-H-Fe] bridging hydrides and two sulfur-bound protons, denoted E 4 (4H), the Janus intermediate, and that breaking the N≡N triple bond requires the reductive elimination (re) of H 2 from E 4 (4H) (6)(7)(8)(9). This process corresponds to the forward direction of the equilibrium in Fig.…”