Abstract
Culmodulin (CaM) binding to neuronal nitric oxide synthase (nNOS) allows NADPII-derived electrons to be transferred from the reductase domain to the oxygenase domain resulting in nitric oxide production. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the reaction between nitric oxide and Superoxide fO: ). we employed the adrenochrome assay to examine whether Ov could be detected during catalytic turnover ol" nNOS in the presence of L-arginine. In order to differentiate between the role played by the reductase domain from that of the oxygenase domain in O," production, we compared nNOS against a nNOS Cys331Ala mutant (CYS331 ). which, as isolated was impaired in the ability to transfer NADPHderived electrons to the heme iron, and against the flavoprotein module of nNOS. We report that O2· - production by nNOS and the CYS-331 mutant is CaM-dependent and that OV production can be modulated by substrates and inhibitors of nNOS. O 2· was also produced by the reductase domain of nNOS; however. it did not display the same CaM dependency. We conclude that both the reduclase and oxygenase domains of nNOS produce O2·-, but that the reductase domain is both necessary and sufficient for O2 production.
| Original language | English (US) |
|---|---|
| Journal | FASEB Journal |
| Volume | 11 |
| Issue number | 9 |
| State | Published - 1997 |
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ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Cell Biology
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Superoxide anion is derived from both the reductase and heme domains of neuronal nitric oxide synthase. / Miller, R. T.; Martasek, P.; Roman, L. J.; Nishimura, J.; Masters, B. S S.
In: FASEB Journal, Vol. 11, No. 9, 1997.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Superoxide anion is derived from both the reductase and heme domains of neuronal nitric oxide synthase
AU - Miller, R. T.
AU - Martasek, P.
AU - Roman, L. J.
AU - Nishimura, J.
AU - Masters, B. S S
PY - 1997
Y1 - 1997
N2 - Culmodulin (CaM) binding to neuronal nitric oxide synthase (nNOS) allows NADPII-derived electrons to be transferred from the reductase domain to the oxygenase domain resulting in nitric oxide production. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the reaction between nitric oxide and Superoxide fO: ). we employed the adrenochrome assay to examine whether Ov could be detected during catalytic turnover ol" nNOS in the presence of L-arginine. In order to differentiate between the role played by the reductase domain from that of the oxygenase domain in O," production, we compared nNOS against a nNOS Cys331Ala mutant (CYS331 ). which, as isolated was impaired in the ability to transfer NADPHderived electrons to the heme iron, and against the flavoprotein module of nNOS. We report that O2· - production by nNOS and the CYS-331 mutant is CaM-dependent and that OV production can be modulated by substrates and inhibitors of nNOS. O 2· was also produced by the reductase domain of nNOS; however. it did not display the same CaM dependency. We conclude that both the reduclase and oxygenase domains of nNOS produce O2·-, but that the reductase domain is both necessary and sufficient for O2 production.
AB - Culmodulin (CaM) binding to neuronal nitric oxide synthase (nNOS) allows NADPII-derived electrons to be transferred from the reductase domain to the oxygenase domain resulting in nitric oxide production. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the reaction between nitric oxide and Superoxide fO: ). we employed the adrenochrome assay to examine whether Ov could be detected during catalytic turnover ol" nNOS in the presence of L-arginine. In order to differentiate between the role played by the reductase domain from that of the oxygenase domain in O," production, we compared nNOS against a nNOS Cys331Ala mutant (CYS331 ). which, as isolated was impaired in the ability to transfer NADPHderived electrons to the heme iron, and against the flavoprotein module of nNOS. We report that O2· - production by nNOS and the CYS-331 mutant is CaM-dependent and that OV production can be modulated by substrates and inhibitors of nNOS. O 2· was also produced by the reductase domain of nNOS; however. it did not display the same CaM dependency. We conclude that both the reduclase and oxygenase domains of nNOS produce O2·-, but that the reductase domain is both necessary and sufficient for O2 production.
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UR - http://www.scopus.com/inward/citedby.url?scp=33750102327&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:33750102327
VL - 11
JO - FASEB Journal
JF - FASEB Journal
SN - 0892-6638
IS - 9
ER -