Inducible nitric-oxide synthase generates superoxide from the reductase domain

Yong Xia, Linda J. Roman, Bettie Sue S Masters, Jay L. Zweier

Research output: Contribution to journalArticlepeer-review

339 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)22635-22639
Number of pages5
JournalJournal of Biological Chemistry
Issue number35
StatePublished - Aug 28 1998

ASJC Scopus subject areas

  • Biochemistry


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