Nitrite and Hydroxylamine as Nitrogenase Substrates: Mechanistic Implications for the Pathway of N2 Reduction
Sudipta Shaw, Dmitriy Lukoyanov, Karamatullah Danyal, Dennis R. Dean, Brian M. Hoffman, and Lance C. Seefeldt
Publication Date (Web): August 19, 2014 (Article)
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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 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.