title: Nitrite and hydroxylamine as nitrogenase substrates: mechanistic implications for the pathway of N₂ reduction.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Shaw
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.forename: Karamatullah
authors.initials: K
authors.lastname: Danyal
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D019811:Hydroxylamine / Q000737:chemistry / Q000378:metabolism*; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism*; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D009573:Nitrites; D019811:Hydroxylamine; D009591:Nitrogenase; D009584:Nitrogen