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

Dmitriy Lukoyanov, Zhi Yong Yang, Brett M. Barney, Dennis R. Dean, Lance C. Seefeldt, Brian M. Hoffman

Research output: Contribution to journalArticlepeer-review

55 Scopus citations


Nitrogenase catalyzes the reduction of N2 and protons to yield two NH 3 and one H 2. Substrate binding occurs at a complex organometallocluster 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 = 1/2 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 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 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 N2 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.

Original languageEnglish (US)
Pages (from-to)5583-5587
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number15
StatePublished - Apr 10 2012


  • EPR
  • Non-Kramers


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