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Cited by 389Nitric Oxide and Endothelin EffectsHemoglobin structure and functionMetal-Catalyzed Oxygenation Mechanisms

Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme.

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Baek, K.J., Thiel, B.A., Lucas, S, Stuehr, D.J.·1993·Journal of Biological Chemistry
DOI 10.1016/s0021-9258(19)36901-7
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Abstract

The cytokine-induced nitric oxide synthase (NOS) of macrophages is a homodimeric enzyme that contains iron protoporphorin IX (heme), FAD, FMN, tetrahydrobiopterin, and calmodulin. To investigate how the enzyme's quaternary structure relates to its catalytic activity and binding of prosthetic groups, dimeric NOS and its subunits were purified separately and their composition and catalytic properties compared. In contrast to dimeric NOS, purified subunits did not synthesize NO or contain bound heme or tetrahydrobiopterin. However, the subunits did contain FAD, FMN, and calmodulin in amounts comparable with dimeric NOS, displayed the light absorbance spectrum of an FAD- and FMN-containing flavoprotein, and generated an air-stable flavin semiquinone radical upon reduction of their ferricyanide-oxidized form. Dimeric NOS and NOS subunits were equivalent in catalyzing electron transfer from NADPH to cytochrome c, dichlorophenolindophenol, or ferricyanide at rates that were 8-30-fold faster than the maximal rate of NO synthesis by dimeric NOS. Reconstitution of subunit NO synthesis required their incubation with L-arginine, tetrahydrobiopterin, and stoichiometric amounts of heme and correlated with formation of a proportional amount of dimeric NOS in all cases. The dimeric NOS reconstituted from its subunits contained 0.9 heme and 0.44 tetrahydrobiopterin bound per subunit and had the spectral and catalytic properties of native dimeric NOS. Thus, NOS subunits are NADPH-dependent reductases that acquire the capacity to synthesize NO only through their dimerization and binding of heme and tetrahydrobiopterin. The ability of heme, tetrahydrobiopterin, and L-arginine to promote subunit dimerization is unprecedented and suggests novel roles for these molecules in forming and stabilizing the active dimeric NOS.

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Authors
Baek, K. J., Thiel, B. A., Lucas, S., & Stuehr, D. J.
Affiliations
Cleveland Clinic (US)
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1993-10
Volume
268
Issue
28
Pages
21120-21129
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/s0021-9258(19)36901-7
OpenAlex
W1558643874
Cited by
389
References
42
Citations by year
2026: 1 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 5 · 2020: 1 · 2019: 6 · 2018: 5 · 2017: 5 · 2016: 8 · 2015: 2 · 2014: 7 · 2013: 11 · 2012: 10
FWCI
18.40
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Cancer Institute (CA-53914)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
Keywords
nitric oxide synthase, macrophages, enzyme subunits, dimerization, heme, tetrahydrobiopterin, flavoprotein, FAD, flavin mononucleotide, calmodulin, L-arginine, NADPH-dependent reductase, enzyme reconstitution, quaternary structure, electron transfer
MeSH terms
Amino Acid Oxidoreductases, Animals, Biopterins, Catalysis, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Flavin-Adenine Dinucleotide, Flavin Mononucleotide, Heme, Macrophages, NADP, Nitric Oxide, Oxidation-Reduction, Oxidoreductases, Substrate Specificity, Nitric Oxide Synthase, Mice

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