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J Biol Chem, Vol. 275, Issue 5, 3201-3205, February 4, 2000

The Ferrous Dioxygen Complex of the Oxygenase Domain of Neuronal Nitric-oxide Synthase^*

Manon Couture, Dennis J. Stuehr^§, and Denis L. Rousseau^¶

From the  Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 and the ^§ Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

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 (Fe²⁺O₂ nNOSoxy). We detect a line at 1135 cm¹ 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 cm¹ with ¹⁸O₂. This line is assigned as the O-O stretching mode (_O-O) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N-hydroxy-L-arginine, in the presence or absence of (6R)-5,6,7,8-tetrahydro-L-biopterin, reveal that the _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.

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