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Inducible nitric-oxide synthase generates superoxide from the reductase domain
Y Xia1, L J Roman, B S Masters
1Molecular and Cellular Biophysics Laboratories, Department of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, Maryland, 21224, USA.
The Journal of Biological Chemistry
|August 26, 1998
Summary
Inducible nitric-oxide synthase (iNOS) generates superoxide (O-2), primarily from its reductase domain. This finding clarifies a key mechanism of iNOS activity and superoxide production.
Area of Science:
- Biochemistry
- Enzymology
- Cellular Biology
Background:
- Neuronal nitric-oxide synthase (nNOS) produces superoxide (O-2) in the absence of L-arginine.
- Previous studies suggested inducible nitric-oxide synthase (iNOS) might also produce O-2 in macrophages, but direct evidence was lacking.
- The mechanism and specific site of O-2 generation by iNOS were unknown.
Purpose of the Study:
- To determine if purified iNOS produces O-2.
- To identify the mechanism and enzyme site responsible for O-2 generation by iNOS.
Main Methods:
- Purified iNOS was analyzed using electron paramagnetic resonance (EPR) spectroscopy with the spin trap 5,5-dimethyl-1-pyrroline N-oxide.
- Experiments involved varying L-arginine concentrations, using superoxide dismutase and catalase, and employing inhibitors like diphenyleneiodonium and cyanide.
- The isolated iNOS reductase domain was also assessed for O-2 generation.
Main Results:
- Prominent O-2 adduct signals were detected from iNOS in the presence of NADPH, confirmed by superoxide dismutase.
- L-arginine significantly reduced O-2 formation, while D-arginine had no effect.
- The flavoprotein inhibitor diphenyleneiodonium blocked O-2 signals, but the heme blocker cyanide did not.
- Direct O-2 generation was observed from the isolated iNOS reductase domain.
Conclusions:
- Inducible nitric-oxide synthase (iNOS) actively generates superoxide (O-2).
- Superoxide production by iNOS primarily occurs at the flavin-binding sites within the reductase domain.
- These findings elucidate a critical aspect of iNOS enzymatic activity and its role in cellular processes.