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*, Lance C. Seefeldt

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)12776-12783
Number of pages8
JournalJournal of the American Chemical Society
Issue number36
StatePublished - Sep 10 2014

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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