Abstract 1685: Overproduction of Oxygen Free Radicals Decrease the Electron Transfer Activity and Protein S-glutathiolation of Mitochondrial Succinate Dehydrogenase in the Postischemic Heart
Mitochondrial superoxide (O2·−) is an important mediator of ischemia/reperfusion (I/R) injury. The O2·− generated in mitochondria acts as a redox signal triggering the cellular event of apoptosis. The succinate dehydrogenase (SDH or Complex II) is one of the major components hosting regulatory thiols in mitochondria. The intrinsic protein S-glutathiolation (PrSSG) at the 70 kDa FAD-binding subunit of SDH was detected in rat heart tissue and isolated SDH using an anti-GSH monoclonal antibody. We have assessed the hypothesis that overproduction of O2·− during I/R injury change the redox status of SDH by decreasing its PrSSG and impairing its electron transfer activity (ETA). When rats are subjected to 45 min of coronary ligation followed by 24 hr reperfusion, the ETA of the SDH in postischemic myocardium was significantly decreased by 41.5±2.85%. No significant difference of the myocardium from I/R and sham control was found in the protein expression of SDH-70 kDa by Western blot. However, the PrSSG at the SDH-70kDa was partially (43.1±5.4%) or completely depleted in the postischemic myocardium obtained from in vivo I/R hearts or the isolated hearts, subjected to global I/R respectively. The above results were further confirmed by using isolated succinate cytochrome c reductase (SCR or Complex II + Complex III) from bovine heart. In the presence of succinate, O2·− generation was catalyzed by SCR as detected by EPR spin-trapping with DEPMPO. Production of O2·− induced oxidative attack of the SDH in SCR, which led to inactivation of its ETA by 60–70%. The gel band of native SDH 70 kDa polypeptide was cut, and digested with trypsin. The digests were then subjected to tandem mass spectrometry (LC/MS/MS) analysis. The results revealed that one specific cysteine residue C90 was involved in the site-specific S-glutathiolation. These results indicate that the GS-binding domain, 77AAFGLSEAGFNTAC90VTK93, is susceptible to redox change induced by oxidative stress. In conclusion, mitochondrial Complex II responds to oxidative stress via redox change of protein S-glutathiolation and decreasing its ETA. The impairment of Complex II with the change of redox modification is unique and useful in probing disease pathogenesis related to ROS-induced mitochondrial dysfunction.