In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O 2 . ). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O 2 . formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O 2 . production does not occur. Moreover, the mechanism and enzyme site

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... le for O 2 . generation is unknown. To determine whether iNOS produces O 2 . and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O 2 . adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O 2 . formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O 2 . signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O 2 . generation. Furthermore, strong O 2 . generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O 2 . , and this mainly occurs at the flavin-binding sites of the reductase domain.