Chen Yeong-Renn, Chen Chwen-Lih, Pfeiffer Douglas R, Zweier Jay L
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.
J Biol Chem. 2007 Nov 9;282(45):32640-54. doi: 10.1074/jbc.M702294200. Epub 2007 Sep 11.
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.
线粒体超氧阴离子（O2.）是缺血/再灌注（I/R）损伤的重要介质。线粒体中产生的O2.还作为触发细胞凋亡的氧化还原信号。琥珀酸泛醌还原酶（SQR或复合体II）是含有调节性硫醇的主要线粒体成分之一。在此，使用抗谷胱甘肽单克隆抗体在大鼠心脏和分离的SQR中检测到SQR 70 kDa FAD结合亚基上的内在蛋白S-谷胱甘肽化（PrSSG）。当大鼠进行30分钟冠状动脉结扎并随后再灌注24小时时，缺血后心肌中SQR的电子传递活性（ETA）显著降低41.5±2.9%。分别从体内局部I/R心脏或分离的整体I/R心脏获得的缺血后心肌中，SQR-70 kDa的PrSSG部分或完全消除。使用分离的琥珀酸细胞色素c还原酶（复合体II + 复合体III）进一步证实了这些结果。在琥珀酸存在下，产生O2.并氧化SCR的SQR部分，导致其ETA降低60 - 70%。切下S-谷胱甘肽化SQR 70 kDa多肽的凝胶条带并用胰蛋白酶消化，消化产物进行液相色谱/串联质谱分析。一个半胱氨酸残基Cys(90)参与了S-谷胱甘肽化。这些结果表明，谷胱甘肽结合结构域（77）AAFGLSEAGFNTACVTK（93）（下划线表示Cys(90)）易受氧化应激诱导的氧化还原变化影响。此外，纯化SQR的体外S-谷胱甘肽化导致SQR衍生的电子传递效率增强，并减少了由O2.诱导的70 kDa衍生蛋白硫自由基的形成。因此，心肌缺血/再灌注期间线粒体复合体II中S-谷胱甘肽化和ETA的降低很明显。复合体II的这种氧化还原触发损伤发生在缺血后心脏，对于识别与活性氧诱导的线粒体功能障碍相关的疾病发病机制应该是有用的。
