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Cited by 86Nitric Oxide and Endothelin EffectsMetal-Catalyzed Oxygenation MechanismsRedox biology and oxidative stress

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·1997·Biochemistry
DOI 10.1021/bi972022c
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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.

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Authors
Miller, R. T., Martásek, P., Roman, L. J., Nishimura, J. S., & Masters, B. S. S.
Affiliations
  • The University of Texas at San Antonio Health Science Center (US)
  • Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
1997-12-01
Volume
36
Issue
49
Pages
15277-15284
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi972022c
OpenAlex
W2005469351
Cited by
86
References
19
Citations by year
2021: 2 · 2019: 1 · 2018: 2 · 2016: 4 · 2015: 3 · 2014: 3 · 2013: 1 · 2012: 3
FWCI
6.71
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Heart, Lung, and Blood Institute (HL30050)
  • National Institute of General Medical Sciences (R01GM052419, GM-52419)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
neuronal nitric oxide synthase, superoxide production, reductase domain, oxygenase domain, calmodulin, peroxynitrite, nitric oxide synthase inhibitors, L-arginine
MeSH terms
Adrenochrome, Calcium, Calmodulin, Cysteine, Epinephrine, Kinetics, Neurons, Oxidoreductases, Superoxides, Mutagenesis, Nitric Oxide Synthase

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