Sulfite Is Not Required for N₂ Reduction Catalyzed by Mo-Nitrogenase

Mo-nitrogenase catalyzes the reduction of dinitrogen (N₂) to two ammonia (NH₃) at the active-site FeMo-cofactor. Substrate activation requires the accumulation of three or four electrons and protons as two Fe-bound hydrides and is coupled to obligatory H₂ release through reductive hydride elimination. Subsequent delivery of four or five additional electrons and protons to the bound N₂ yields two NH₃ molecules. Increasing evidence suggests that at least one belt sulfide within FeMo-cofactor is dynamically involved in the catalytic cycle. A recent report further proposed that sulfite (SO₃^2-) is required for N₂ reduction, with sulfite binding required for NH₃ release and a subsequent six-electron reduction of the bound sulfite to regenerate the resting cofactor. To test this proposal, we conducted turnover studies of Mo-nitrogenase under sulfite-free conditions using a reduced viologen as reductant and protein preparations devoid of dithionite or sulfite. Under these conditions, nitrogenase effectively catalyzed both N₂ reduction and proton reduction, exhibiting steady-state turnover under N₂ for 6 min, with a turnover number exceeding 150, approaching that observed with dithionite as reductant. The same H₂-formed/N₂-reduced ratio was observed whether dithionite or the viologen species was used as reductant. Further, EPR spectroscopic analyses showed that the FeMo-cofactor returned to its resting state after multiple catalytic cycles in the absence of sulfite. Finally, physiological bypass of sulfite formation does not affect the capacity for diazotrophic growth of the model nitrogen-fixing organism Azotobacter vinelandii. These results demonstrate that sulfite is not required for Mo-nitrogenase-catalyzed N₂ reduction either in vitro or in vivo.