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

Nitrogenase catalyzes the reduction of N(2) and protons to yield two NH(3) and one H(2). 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-CH(3)), or hydrazine as substrate recently was shown to trap a common S = ½ intermediate, designated I. It was further concluded that the two N-atoms of N(2) are hydrogenated alternately ("Alternating" (A) pathway). In the present work, Q-band CW EPR and (95)Mo 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 (14,15)N isotopologs plus (1,2)H isotopologs of methyldiazene. It is concluded that: H has NH(2) bound to FeMo-co and corresponds to the penultimate intermediate of N(2) hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH(3); I corresponds to the final intermediate in N(2) reduction, the state formed after accumulation of eight electrons/protons, with NH(3) 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 N(2) reduction by nitrogenase.