Abstract 219: Oxidative Modification with Protein Tyrosine Nitration Occurs following Deglutathiolation of the 70 kDa Flavoprotein of Mitochondrial Complex II Is Associated with Loss of Electron Transfer Activity in the Post-Ischemic Myocardium
Increased O2•− and NO production is a key mechanism of mitochondrial dysfunction in ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment (42% reduction of electron transfer activity (ETA)) and decreased protein S-glutathiolation of the 70 kDa flavoprotein occurs in the post-ischemic heart. To test the hypothesis that oxidative modification with protein tyrosine nitration is expected to follow deglutathiolation of the SQR 70 kDa subunit in the post-ischemic myocardium, we used the identified S-glutathiolated peptide (77AAFGLSEAGFNTAC90VTK93) 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 and high specificity to isolated SQR and the SQR in myocardial tissue. Antibodies of AbGSC90 moderately inhibited the electron transfer (~25% inhibition) and superoxide generation (~26% inhibition) activities of SQR, implicating the GS-binding domain in the catalytic functions of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 hours of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90, and probed with antibodies against 3-nitrotyrosine (Ab3NT). Enhancement of protein nitration was detected in the post-ischemic myocardium. Immunofluorescence confocal microscopy with AbGSC90 and Ab3NT further revealed significant and intense 3-nitrotyrosine staining localization in the SQR 70 kDa protein. 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 (~65% ETA remained). Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis indicated that Y56 and Y142 were involved in protein nitration. When the isolated SQR was subjected to in vitro S-glutathiolation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased (~97% ETA remained) thus confirming the protective effect of S-glutathiolation against the oxidative damage of nitration.