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 = 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).