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 (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.