Freeze-quenching nitrogenase during turnover with N-2 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 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 degrees C 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 NH3.