The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase.
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 (Fe(2+)O(2) 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 (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(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.
Couture
M
M
Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Stuehr
D J
DJ
Rousseau
D L
DL
D000818:Animals; D004926:Escherichia coli; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D005296:Ferrous Compounds; D009419:Nerve Tissue Proteins; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen