Peroxynitrite-Mediated Oxidative Modifications of Complex II: Relevance in Myocardial Infarction

Peroxynitrite-Mediated Oxidative Modifications of Complex II: Relevance in Myocardial Infarction

Zhang L W; Chen C L; Kang P T; Garg V; Hu K L; Green-Church K B; Chen Y R

Biochemistry

2010

2010-03

Increased O-2(center dot-) and NO production is a key mechanism of mitochondrial dysfunction in mycocardial ischemia/reperfusion injury. In complex impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO-) ill vivo [Chen, Y.-R., et ill. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved ill OONO--mediated Oxidative post-translational modifications relevant ill myocardial infarction, We subjected Isolated myocardial complex 11 to ill vltro protein nitration with OONO-. This resulted ill site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa POlyPePtldC MIS subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis Nitration Of Y-56 and Y-142 was previosly reported. Further analysis revealed that C-267, C-476 and C-537 are involved ill OONO--mediated S-sulfonation. To identify the disulfide formation mediated by OONO-, nitrated complex 11 was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C-306-C-312,C-439-C-444, and C-288-C-575, were involved in OONO--mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS Was used to define Oxidative modification with protein radical formation. An OONO--dependent DMPO adduct was detected. and further LC-MS/MS analysis indicated C-288 and C-655 were involved in DMPO binding. These I-CSLIltS offered a complete profile of OONO--mediated Oxidative modifications that may be relevant Ill the disease model of myocardial infarction.

Biochemistry & Molecular Biology; cytochrome-c; fatty-acids; identification; membrane; mitochondrial; modulation; nitric-oxide; postischemic heart; succinate-ubiquinone; thiyl

Journal Article

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2529-2539

11

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