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 (Fe2+O2 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 O-18(2). This line is assigned as the O-O stretching mode (upsilon(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 upsilon(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.