Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂.
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.
‡Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.
Dmitriy
D
Lukoyanov
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States.
Zhi-Yong
ZY
Yang
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States.
Nimesh
N
Khadka
§Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.
Dennis R
DR
Dean
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States.
Lance C
LC
Seefeldt
‡Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.
Brian M
BM
Hoffman
D055162:Biocatalysis; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D003067:Coenzymes; D006859:Hydrogen; D009591:Nitrogenase; D009584:Nitrogen