Nitriteand Hydroxylamine as Nitrogenase Substrates:Mechanistic Implications for the Pathway of N2 Reduction

摘要

Investigations of reduction of nitrite (NO2^–) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2^– + 6e^– + 12ATP + 7H⁺ → NH3 + 2H2O + 12ADP + 12Pi. 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 resultspresented here, combined with the parallels with ccNIR, support aN₂ fixation mechanism in which liberation of the firstNH₃ occurs upon delivery of five [e^–/H⁺] to N₂, but a total of seven [e^–/H⁺] to FeMo-co when obligate H₂ evolutionis considered, and not earlier in the reduction process.