title: Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase.
abstract: 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.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Yang
authors.forename: Brett M
authors.initials: BM
authors.lastname: Barney
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D055162:Biocatalysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D009584:Nitrogen / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013113:Spin Labels
chemical_list: D013113:Spin Labels; D009591:Nitrogenase; D009584:Nitrogen