Nitrite and Hydroxylamine as Nitrogenase Substrates: Mechanistic Implications for the Pathway of N(2) Reduction

[Image: see text] Investigations of reduction of nitrite (NO(2)(–)) to ammonia (NH(3)) by nitrogenase indicate a limiting stoichiometry, NO(2)(–) + 6e(–) + 12ATP + 7H(+) → NH(3) + 2H(2)O + 12ADP + 12P(i). Two intermediates freeze-trapped during NO(2)(–) turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N(2) reduction. The proposed NO(2)(–) reduction intermediate hydroxylamine (NH(2)OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N(2) and NO(2)(–) reductions in light of their common reduction intermediates and of NO(2)(–) reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO(2)(–) reduction by nitrogenase begins with the generation of NO(2)H bound to a state in which the active-site FeMo-co (M) has accumulated two [e(–)/H(+)] (E(2)), stored as a (bridging) hydride and proton. Proton transfer to NO(2)H and H(2)O loss leaves M–[NO(+)]; transfer of the E(2) 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 N(2) and NO(2)(–) reduction pathways converge upon reduction of NH(2)NH(2) and NH(2)OH bound states to form state H with [−NH(2)] bound to M. Final reduction converts H to I, with NH(3) bound to M. The results presented here, combined with the parallels with ccNIR, support a N(2) fixation mechanism in which liberation of the first NH(3) occurs upon delivery of five [e(–)/H(+)] to N(2), but a total of seven [e(–)/H(+)] to FeMo-co when obligate H(2) evolution is considered, and not earlier in the reduction process.