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 (OX). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2(radical anion) 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(radical anion) production does not occur. Moreover, the mechanism and enzyme site responsible for OX generation is unknown. To determine whether iNOS produces O-2(radical anion) 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(radical anion) adduct signals were detected from NOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this OX formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2(radical anion) signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on OX generation. Furthermore, strong O-2(radical anion) generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2(radical anion), and this mainly occurs at the flavin-binding sites of the reductase domain.