Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism to Achieve N₂ Reduction

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N₂ reduction to 2NH₃: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe₇). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N₂ binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E₄(4H) ("Janus") state. N₂ binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H₂, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N₂/D₂. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H₂ formation by hydride protonolysis (k_HP) but significant differences in the rate constant for H₂ re with N₂ binding/reduction (k_re). We now report that V-nitrogenase also exhibits HD formation during N₂/D₂ turnover (and H₂ inhibition of N₂ reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N₂ binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (k_HP) for H₂ production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (k_re) for the re of H₂ coupled to N₂ binding/reduction, which decreases in the order Mo > V > Fe.