Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption

Fabio V. Fonseca, Kandasamy Ravi, Dean Wiseman, Monorama Tummala, Cynthia Harmon, Victor Ryzhov, Jeffrey R. Fineman, Stephen Matthew Black

Research output: Contribution to journalArticle

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Abstract

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H2O2), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H 2O2 inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H2O2. Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H2O2 caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H2O2 increased the oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2), whereas preincubation of heNOS with excess BH4 prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH4 is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H2O2 could also induce dimer collapse and that increasing cellular BH4 levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H2O2. This study links the inhibitory action of H 2O2 on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)149-160
Number of pages12
JournalDNA and cell biology
Volume29
Issue number3
DOIs
StatePublished - Mar 1 2010

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Nitric Oxide Synthase Type III
Hydrogen Peroxide
Zinc
Mass Spectrometry
Metals
Binding Sites
Cysteine
Cysteic Acid
Hydrogen Bonding
Catalase
Sheep
Endothelial Cells

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

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Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption. / Fonseca, Fabio V.; Ravi, Kandasamy; Wiseman, Dean; Tummala, Monorama; Harmon, Cynthia; Ryzhov, Victor; Fineman, Jeffrey R.; Black, Stephen Matthew.

In: DNA and cell biology, Vol. 29, No. 3, 01.03.2010, p. 149-160.

Research output: Contribution to journalArticle

Fonseca, Fabio V. ; Ravi, Kandasamy ; Wiseman, Dean ; Tummala, Monorama ; Harmon, Cynthia ; Ryzhov, Victor ; Fineman, Jeffrey R. ; Black, Stephen Matthew. / Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption. In: DNA and cell biology. 2010 ; Vol. 29, No. 3. pp. 149-160.
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