Unification of reaction pathway and kinetic scheme for N^sub 2^ reduction catalyzed by nitrogenase

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