Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production

R. Timothy Miller, Pavel Martásek, Linda J. Roman, Jonathan S. Nishimura, Bettie Sue Siler Masters

Research output: Contribution to journalArticle

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Abstract

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CAM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2 .-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2 .- during catalytic turnover in the presence of L- arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2 .-production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2 .- production by nNOS and the CYS-331 mutant is CAM-dependent and that O2 .- production can be modulated by substrates and inhibitors of nNOS. O2 .- was also produced by the reductase domain of nNOS; however, it did not display the same CAM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2 .-, but that the reductase domain is both necessary and sufficient for O2 .- production.

Original languageEnglish (US)
Pages (from-to)15277-15284
Number of pages8
JournalBiochemistry
Volume36
Issue number49
DOIs
StatePublished - Dec 9 1997

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Nitric Oxide Synthase Type I
Superoxides
Oxidoreductases
Oxygenases
Calmodulin
Peroxynitrous Acid
Heme
NADP
Arginine
Nitric Oxide
Adrenochrome
Electrons
Nitrogen Oxides
Heme Oxygenase (Decyclizing)
Flavoproteins
Citrulline
Guanylate Cyclase
Assays
Iron
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Miller, R. T., Martásek, P., Roman, L. J., Nishimura, J. S., & Masters, B. S. S. (1997). Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production. Biochemistry, 36(49), 15277-15284. https://doi.org/10.1021/bi972022c

Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production. / Miller, R. Timothy; Martásek, Pavel; Roman, Linda J.; Nishimura, Jonathan S.; Masters, Bettie Sue Siler.

In: Biochemistry, Vol. 36, No. 49, 09.12.1997, p. 15277-15284.

Research output: Contribution to journalArticle

Miller, RT, Martásek, P, Roman, LJ, Nishimura, JS & Masters, BSS 1997, 'Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production', Biochemistry, vol. 36, no. 49, pp. 15277-15284. https://doi.org/10.1021/bi972022c
Miller, R. Timothy ; Martásek, Pavel ; Roman, Linda J. ; Nishimura, Jonathan S. ; Masters, Bettie Sue Siler. / Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production. In: Biochemistry. 1997 ; Vol. 36, No. 49. pp. 15277-15284.
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