Nitrite and hydroxylamine as nitrogenase substrates: Mechanistic implications for the pathway of N₂ reduction

Investigations of reduction of nitrite (NO₂^-) to ammonia (NH₃) by nitrogenase indicate a limiting stoichiometry, NO₂^- + 6e^- + 12ATP + 7H⁺ → NH₃ + 2H₂O + 12ADP + 12P_i. Two intermediates freeze-trapped during NO₂^- turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N₂ reduction. The proposed NO₂^- reduction intermediate hydroxylamine (NH₂OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N₂ and NO₂^- reductions in light of their common reduction intermediates and of NO₂^- reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO₂^- reduction by nitrogenase begins with the generation of NO₂H bound to a state in which the active-site FeMo-co (M) has accumulated two [e^-/H⁺] (E₂), stored as a (bridging) hydride and proton. Proton transfer to NO₂H and H₂O loss leaves M-[NO⁺]; transfer of the E₂ hydride to the [NO⁺] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M-[NO] thermodynamic "sink". The N₂ and NO₂^- reduction pathways converge upon reduction of NH₂NH₂ and NH₂OH bound states to form state H with [-NH₂] bound to M. Final reduction converts H to I, with NH₃ bound to M. The results presented here, combined with the parallels with ccNIR, support a N₂ fixation mechanism in which liberation of the first NH₃ occurs upon delivery of five [e^-/H⁺] to N₂, but a total of seven [e^-/H⁺] to FeMo-co when obligate H₂ evolution is considered, and not earlier in the reduction process.