Mitochondrial complex II in the post-ischemic heart - Oxidative injury and the role of protein S-glutathionylation

Mitochondrial superoxide (O-2(radical-anion)) is an important mediator of ischemia/ reperfusion ( I/ R) injury. The O-2(radical-anion) generated in mitochondria also acts as a redox signal triggering cellular apoptosis. The enzyme succinate ubiquinone reductase ( SQR or complex II) is one of the major mitochondrial components hosting regulatory thiols. Here the intrinsic protein S- glutathionylation ( PrSSG) at the 70- kDa FAD- binding subunit of SQR was detected in rat heart and in isolated SQR using an anti- GSH monoclonal antibody. When rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion, the electron transfer activity ( ETA) of SQR in post- ischemic myocardium was significantly decreased by 41.5 +/- 2.9%. The PrSSGs of SQR- 70 kDa were partially or completely eliminated in post- ischemic myocardium obtained from in vivo regional I/ R hearts or isolated global I/ R hearts, respectively. These results were further confirmed by using isolated succinate cytochrome c reductase ( complex II + complex III). In the presence of succinate, O-2(radical-anion) was generated and oxidized the SQR portion of SCR, leading to a 60 - 70% decrease in its ETA. The gel band of the S- glutathionylated SQR 70- kDa polypeptide was cut out and digested with trypsin, and the digests were subjected to liquid chromatography/ tandem mass spectrometry analysis. One cysteine residue, Cys(90), was involved in S- glutathionylation. These results indicate that the glutathione-binding domain, (77)AAFGLSEAGFNTACVTK(93) ( where underline indicates Cys90), is susceptible to redox change induced by oxidative stress. Furthermore, in vitro S- glutathionylation of purified SQR resulted in enhanced SQR- derived electron transfer efficiency and decreased formation of the 70- kDa- derived protein thiyl radical induced by O-2(radical-anion).Thus, the decreasing S- glutathionylation and ETA in mitochondrial complex II are marked during myocardial ischemia/ reperfusion. This redox- triggered impairment of complex II occurs in the post- ischemic heart and should be useful to identify disease pathogenesis related to reactive oxygen species- induced mitochondrial dysfunction.