The ferrous dioxygen complex of the oxygenase domain of neuronal nitric- oxide synthase

Manon Couture, Dennis J. Stuehr, Denis L. Rousseau

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57 Scopus citations


The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe2O2 nNOSoxy). We detect a line at 1135 cm-1 in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm-1 with 18O2. This line is assigned as the O-O stretching mode (V(o-o)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or Nω-hydroxy- L-arginine, in the presence or absence of (6R)-5,6,7,8-tetrahydro-L- biopterin, reveal that the V(o-o) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.

Original languageEnglish (US)
Pages (from-to)3201-3205
Number of pages5
JournalJournal of Biological Chemistry
Issue number5
StatePublished - Feb 4 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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