Mechanism of substrate inhibition in cytochrome-c dependent NO reductases from denitrifying bacteria (cNORs)

Hirotoshi Matsumura, Abayomi S. Faponle, Peter Leon Hagedoorn, Takehiko Tosha, Sam P. de Visser, Pierre Moënne-Loccoz

Research output: Contribution to journalArticlepeer-review

Abstract

Steady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 μM, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm−1 that downshifts with 15NO and 15N18O is assigned to a ν(NO2) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations reproduces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173–5186] that proposed substrate inhibition through a radical combination reaction between the diferric μ-oxo group and an NO molecule to form a heme Fe(III)-nitrito-FeB(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N2O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric μ-oxo group and N2O without protonation of the putative hyponitrite intermediate.

Original languageEnglish (US)
Article number111781
JournalJournal of Inorganic Biochemistry
Volume231
DOIs
StatePublished - Jun 2022
Externally publishedYes

Keywords

  • Iron proteins
  • Nitric oxide reductases
  • Reaction mechanisms
  • Spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

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