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 N-2 reduction to 2NH(3): 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 N-2 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 E-4(4H) (Janus) state. N-2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H-2, 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 N-2/D-2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H-2 formation by hydride protonolysis (k(HP)) but significant differences in the rate constant for H-2 re with N-2 binding/reduction (k(re)). We now report that V-nitrogenase also exhibits HD formation during N-2/D-2 turnover (and H-2 inhibition of N-2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N-2 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 H-2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (k(re)) for the re of H-2 coupled to N-2 binding/reduction, which decreases in the order Mo > V > Fe.