Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N2 for H2

Dmitriy Lukoyanov, Zhi Yong Yang, Nimesh Khadka, Dennis R. Dean*, Lance C. Seefeldt, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticle

66 Scopus citations

Abstract

Freeze-quenching nitrogenase during turnover with N2 traps an S = 1/2 intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e-/H+] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3. (Chemical Equation Presented).

Original languageEnglish (US)
Pages (from-to)3610-3615
Number of pages6
JournalJournal of the American Chemical Society
Volume137
Issue number10
DOIs
StatePublished - Mar 18 2015

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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