title: Protein tyrosine nitration of the flavin subunit is associated with oxidative modification of mitochondrial complex II in the post-ischemic myocardium.
abstract: Increased O(2)* 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 ((77)AAFGLSEAGFNTACVTK(93)) 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.
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine.
authors.forename: Chwen-Lih
authors.initials: CL
authors.lastname: Chen
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine.
authors.forename: Jingfeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Sharad
authors.initials: S
authors.lastname: Rawale
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine.
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Pravin P T
authors.initials: PPT
authors.lastname: Kaumaya
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine; Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210.
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine; Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210. Electronic address: yeongrenn.chen@osumc.edu.
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
mesh_terms: D000818:Animals; D002417:Cattle; D042963:Electron Transport Complex II / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D042963:Electron Transport Complex II