Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation.
Mitochondrial superoxide (O2.) is an important mediator of ischemia/reperfusion (I/R) injury. The O2. 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, O2. 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 Cys(90)), 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 O2. . 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.
Chen
Yeong-Renn
YR
Division of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, 473 W. 12th Avenue, Columbus, OH 43210, USA. yeong-renn.chen@osumc.edu
Chen
Chwen-Lih
CL
Pfeiffer
Douglas R
DR
Zweier
Jay L
JL
D000595:Amino Acid Sequence; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D042963:Electron Transport Complex II / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017202:Myocardial Ischemia / Q000378:metabolism*; D018384:Oxidative Stress; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D005415:Flavins; D005609:Free Radicals; D021122:Protein Subunits; D013481:Superoxides; D042963:Electron Transport Complex II; D005978:Glutathione