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 (SO32-) 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.
Yang et al. (2026) studied this question.