Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase

Nitrogenase catalyzes the reduction of N₂ and protons to yield two NH₃ and one H₂. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH₃), or hydrazine as substrate recently was shown to trap a common intermediate, designated I. It was further concluded that the two N-atoms of N₂ are hydrogenated alternately (“Alternating” (A) pathway). In the present work, Q-band CW EPR and ⁹⁵Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of ^14,15N isotopologs plus ^1,2H isotopologs of methyldiazene. It is concluded that: H has NH₂ bound to FeMo-co and corresponds to the penultimate intermediate of N₂ hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH₃; I corresponds to the final intermediate in N₂ reduction, the state formed after accumulation of eight electrons/protons, with NH₃ still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N₂ reduction by nitrogenase.