Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

Jin Qian, Feng Chen, Yevgeniy Kovalenkov, Deepesh Pandey, M. Arthur Moseley, Matthew W. Foster, Stephen M. Black, Richard C. Venema, David W. Stepp, David J.R. Fulton

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Abstract

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.

Original languageEnglish (US)
Pages (from-to)1806-1819
Number of pages14
JournalFree Radical Biology and Medicine
Volume52
Issue number9
DOIs
Publication statusPublished - May 1 2012

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Keywords

  • Free radicals
  • NADPH oxidase
  • Nitric oxide
  • Nox5
  • Reactive oxygen species
  • S-nitrosylation

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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