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Cited by 59Nitric Oxide and Endothelin EffectsMetal-Catalyzed Oxygenation MechanismsHemoglobin structure and function

Analysis of Neuronal NO Synthase under Single-Turnover Conditions: Conversion of Nω-Hydroxyarginine to Nitric Oxide and Citrulline

Abu-Soud, Husam M., Presta, Anthony, Mayer, Bernd, Stuehr, Dennis J.·1997·Biochemistry
DOI 10.1021/bi971414g
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Abstract

Nitric oxide synthases (NOSs) are proposed to generate NO and citrulline from L-arginine in two steps: initial N-hydroxylation to generate Nomega-hydroxyarginine (NOHA) followed by a three-electron oxidation of the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation of O2. Studies done under multiple-turnover conditions suggest that 0.5 mol of NADPH is consumed to convert 1 mol of NOHA to products, implying that one electron from NADPH may be sufficient. To test this, we studied NOHA oxidation under single-turnover conditions using neuronal NOS (nNOS), whose heme iron reduction requires bound calmodulin. The heme iron in calmodulin-bound nNOS was reduced with excess NADPH under anaerobic conditions, calmodulin was then dissociated from nNOS to prevent subsequent heme iron reduction, NOHA was added, and the reaction initiated by exposure to air. Spectra obtained at each step were consistent with buildup of NOHA-bound ferrous nNOS prior to air exposure. Reactions containing graded amounts of nNOS produced L-citrulline in linear relation (1.2 +/- 0.1 mol of citrulline per mole of nNOS). Nitrite and nitrate also accumulated as NO-derived products. Control reactions that contained L-arginine instead of NOHA, no enzyme, or ferric nNOS did not generate products. Thus supplying a single electron from NADPH to the heme iron permits nNOS to catalyze one full round of citrulline and NO synthesis from NOHA upon exposure to O2. These data provide a molecular explanation for the NADPH requirement in the second step of the biosynthetic reaction, implicate ferrous-dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.

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  • The ratio of NADPH to l-citrulline is close to the optimal value of 1.5 molecules of NADPH per l-citrulline (and NO) generated.

    “The electrons transferred from NADPH are almost quantitatively used to catalyze l-arginine oxidation, and the ratio of NADPH to l-citrulline is close to the optimal value of 1.5 molecules of NADPH per l-citrulline (and NO) generated (3, 590).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [3]

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Authors
Abu-Soud, H. M., Presta, A., Mayer, B., & Stuehr, D. J.
Affiliations
  • Department of Immunology, Cleveland Clinic Research Institute, Cleveland, Ohio 44195, and the Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria
  • University of Graz (AT)
Corresponding author
Stuehr, Dennis J.
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
1997-09-01
Volume
36
Issue
36
Pages
10811-10816
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi971414g
OpenAlex
W1977923059
Cited by
59
References
13
Citations by year
2021: 1 · 2018: 2 · 2017: 1 · 2016: 2 · 2014: 3 · 2013: 1 · 2012: 1
FWCI
4.88
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (GM51491, R01GM051491)
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 › Cell Biology › Hemoglobin structure and function
Keywords
neuronal nitric oxide synthase, nitric oxide synthase, nitric oxide, citrulline, single-turnover kinetics, NADPH, heme iron reduction, calmodulin, L-arginine, oxygen activation
MeSH terms
Animals, Arginine, Citrulline, Nerve Tissue Proteins, Nitric Oxide, Nitric Oxide Synthase

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