Inducible nitric-oxide synthase generates superoxide from the reductase domain

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O₂^.-). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O₂/^.- 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 O₂/^.- production does not occur. Moreover, the mechanism and enzyme site responsible for O₂/^.- generation is unknown. To determine whether iNOS produces O₂/^.- 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 O₂/^.- 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 O₂/^.- formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O₂/^.- signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O₂/^.- generation. Furthermore, strong O₂/^.- generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O₂/^.- and this mainly occurs at the flavin-binding sites of the reductase domain.