Time-Resolved EPR Study of H₂ Reductive Elimination from the Photoexcited Nitrogenase Janus E₄(4H) Intermediate

Nitrogenase is activated for N₂ reduction through the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co: Fe7S9MoC; homocitrate) to form the key Janus intermediate, denoted E₄(4H), whose lowest-energy structure contains two [Fe-H-Fe] bridging hydrides and two protons bound to the sulfurs that also bridge the Fe pairs. In the critical step of catalysis, a H₂ complex transiently produced by reductive elimination (re) of the hydrides of E₄(4H), denoted E₄(H₂;2H), undergoes H₂ displacement by N₂, which then undergoes the otherwise energetically unfavorable cleavage of the NN triple bond. In pursuing the study of the re activation process, we have employed a photochemical approach to obtaining its atomic-level details. Continuous 450 nm irradiation of the ground state of the dihydride Janus intermediate, denoted E₄(4H)^a, in an EPR cavity at cryogenic temperatures causes photoinduced re of H₂ to generate E₄(H₂;2H). We here extend this photochemical approach with time-resolved EPR studies of the photolysis process on the ns time scale. These studies reveal an additional intermediate in the catalytic reductive elimination process, an isomer of the E₄(4H) FeMo-co metal-ion core that is formed prior to E₄(H₂;2H) and is thought to be created by breaking an Fe-SH bond, thus further integrating the calculational and structural studies into the experimentally determined mechanism by which nitrogenase is activated to cleave the NN triple bond.