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 (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 responsible 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.
Molecular and Cellular Biophysics Laboratories, Department of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, Maryland, 21224, USA.
Y
Y
Xia
L J
LJ
Roman
B S
BS
Masters
J L
JL
Zweier
D000818:Animals; D002384:Catalysis; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D051379:Mice; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010088:Oxidoreductases / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013113:Spin Labels; D013481:Superoxides / Q000378:metabolism*
D003497:Cyclic N-Oxides; D011994:Recombinant Proteins; D013113:Spin Labels; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse