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

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• 作者：Liwen Zhang ; Chwen-Lih Chen ; Patrick T. Kang ; Vivek Garg ; Keli Hu ; Kari B. Green-Church ; Yeong-Renn Chen
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：March 23, 2010
• 年：2010
• 卷：49
• 期：11
• 页码：2529-2539
• 全文大小：1210K
• 年卷期：v.49,no.11(March 23, 2010)
• ISSN：1520-4995

文摘

Increased O₂^•− and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative 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⁻) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991−28003]. To gain deeper insights into the redox protein thiols involved in OONO⁻-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO⁻. This resulted in 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 polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC−MS/MS analysis. Nitration of Y₅₆ and Y₁₄₂ was previously reported. Further analysis revealed that C₂₆₇, C₄₇₆, and C₅₃₇ are involved in OONO⁻-mediated S-sulfonation. To identify the disulfide formation mediated by OONO⁻, nitrated complex II 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, C₃₀₆−C₃₁₂, C₄₃₉−C₄₄₄, and C₂₈₈−C₅₇₅, 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 C₂₈₈ and C₆₅₅ were involved in DMPO binding. These results offered a complete profile of OONO⁻-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.