Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium

Chwen Lih Chen, Jingfeng Chen, Sharad Rawale, Saradhadevi Varadharaj, Pravin P T Kaumaya, Jay L. Zweier, Yeong Renn Chen

Research output: Contribution to journalArticle

Abstract

Increased O2.- and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart (Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide (77AAFGLSEAGFNTACVTK93) of the 70-kDa protein as a chimeric epitope incorporating a "promiscuous" T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.

Original languageEnglish (US)
Pages (from-to)27991-28003
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number41
DOIs
StatePublished - Oct 10 2008
Externally publishedYes

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Nitration
Tyrosine
Myocardium
Proteins
Peroxynitrous Acid
Antibodies
Electron Transport Complex II
Electrons
Myocardial Reperfusion Injury
4,6-dinitro-o-cresol
Peptides
T-Lymphocyte Epitopes
Electron Transport Complex III
Liquid chromatography
Electron Transport
Tandem Mass Spectrometry
Reperfusion Injury
Superoxides
Liquid Chromatography
Reperfusion

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium. / Chen, Chwen Lih; Chen, Jingfeng; Rawale, Sharad; Varadharaj, Saradhadevi; Kaumaya, Pravin P T; Zweier, Jay L.; Chen, Yeong Renn.

In: Journal of Biological Chemistry, Vol. 283, No. 41, 10.10.2008, p. 27991-28003.

Research output: Contribution to journalArticle

Chen, Chwen Lih ; Chen, Jingfeng ; Rawale, Sharad ; Varadharaj, Saradhadevi ; Kaumaya, Pravin P T ; Zweier, Jay L. ; Chen, Yeong Renn. / Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 41. pp. 27991-28003.
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AU - Chen, Jingfeng

AU - Rawale, Sharad

AU - Varadharaj, Saradhadevi

AU - Kaumaya, Pravin P T

AU - Zweier, Jay L.

AU - Chen, Yeong Renn

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