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


  • 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.

published proceedings

  • J Am Chem Soc

altmetric score

  • 0.5

author list (cited authors)

  • Lukoyanov, D., Yang, Z., Khadka, N., Dean, D. R., Seefeldt, L. C., & Hoffman, B. M

citation count

  • 80

complete list of authors

  • Lukoyanov, Dmitriy||Yang, Zhi-Yong||Khadka, Nimesh||Dean, Dennis R||Seefeldt, Lance C||Hoffman, Brian M

publication date

  • March 2015