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J Am Chem Soc. 2015 Mar 18;137(10):3610-5. doi: 10.1021/jacs.5b00103. Epub 2015 Mar 5.

Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂.

Author information

1
‡Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.
2
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States.
3
§Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.

Abstract

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ 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.

PMID:
25741750
PMCID:
PMC4374740
DOI:
10.1021/jacs.5b00103
[Indexed for MEDLINE]
Free PMC Article

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