Nitrite and hydroxylamine as nitrogenase substrates: mechanistic implications for the pathway of Nā‚‚ reduction. Academic Article uri icon


  • Investigations of reduction of nitrite (NO2(-)) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2(-) + 6e(-) + 12ATP + 7H(+) ā†’ 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.

published proceedings

  • J Am Chem Soc

altmetric score

  • 0.75

author list (cited authors)

  • Shaw, S., Lukoyanov, D., Danyal, K., Dean, D. R., Hoffman, B. M., & Seefeldt, L. C

citation count

  • 30

complete list of authors

  • Shaw, Sudipta||Lukoyanov, Dmitriy||Danyal, Karamatullah||Dean, Dennis R||Hoffman, Brian M||Seefeldt, Lance C

publication date

  • September 2014