Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N_2 for H_2

摘要

Freeze-quenching nitrogenase during turnover with N_2 traps an S =1/2 intermediate that was shown by ENDOR and EPR spectroscopy to contain N_2 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 E_0 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 ℃ cryoannealing of EG prepared under turnover conditions in which the concentrations of N_2 and H_2 ([H_2], [N_2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H_2] and slowed by increasing [N_2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H_2] and/or high [N_2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H_2 with concomitant N_2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N_2 and two [e~-/ H~+] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N_2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N_2 and H_2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N_2 to two NH_3.