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

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

Research output: Contribution to journalArticle

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Abstract

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
Volume275
Issue number5
DOIs
StatePublished - Feb 4 2000

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Oxygenases
Nitric Oxide Synthase Type I
Oxygen
Arginine
Biopterin
Pterins
Raman Spectrum Analysis
Substrates
Heme
Nitric Oxide Synthase
Stretching
Raman spectroscopy
Raman scattering
Catalytic Domain
Nitric Oxide
Wavelength
Enzymes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

The ferrous dioxygen complex of the oxygenase domain of neuronal nitric- oxide synthase. / Couture, Manon; Stuehr, Dennis J.; Rousseau, Denis L.

In: Journal of Biological Chemistry, Vol. 275, No. 5, 04.02.2000, p. 3201-3205.

Research output: Contribution to journalArticle

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