Mitochondria are the major cellular source of oxygen free radicals (1, 2). The generation of reactive oxygen species (ROS) 2 and free radical(s) in mitochondria is particularly relevant under the physiological conditions of low oxygen tension such as state 4 respiration or certain pathological conditions such as inflammation and ischemia-reperfusion injury (3-5). To understand disease processes associated with oxidative stress, it is necessary to understand the fundamental mechanisms of mitochondrial-derived oxygen-free radical generation. A decrease in the rate of mitochondrial oxidative phosphorylation can increase the production of superoxide anion radical (O 2 . ) in the early stages of the electron transport chain (ETC). Two segments of the ETC have been widely hypothesized to be responsible for O 2 . generation in mitochondria (6). One site is located on complex III. At this site, O 2 .production is mediated through the Q-cycle mechanism, in which electron leakage results from the auto-oxidation of ubisemiquinone and reduced cytochrome b 566 (7-10). The other site is located on complex I (11). Purified bovine heart complex I (or NADH ubiquinone reductase) contains up to 46 different subunits with a total molecular mass of 980 kDa (12). With the use of chaotropic anions such as perchlorate, complex I can be resolved into three fractions: a flavoprotein fraction (Fp), an iron-sulfur (Fe-S) protein fraction (Ip), and a hydrophobic protein fraction (Hp). The redox centers of complex I that are involved in mediation of two-electron transfer from NADH to ubiquinone include a non-covalent binding FMN, and ubiquinone (14,15).In addition to the function of electron transfer required for energy transduction in mitochondria, complex I is also responsible for O 2 . generation, which is frequently linked to disease pathophysiology (16). The oxidative damage of complex I has been identified in a number of diseased conditions such as ischemia-reperfusion injury (17)(18)(19) and Parkinson's disease (20,21). Two redox components of complex I are logically hypothesized to be involved in the electron leakage for O 2. generation: one located on the FMN-containing Fp fraction, and one on the Q-binding site that mediates ubiquinone reduction. Based on this hypothesis, reduced FMN semiquinone radical (FMNH ⅐ ) (22) and ubisemiquinone radical (Q ៛ ) can provide the sources of O 2 . generation.The Fp fraction of complex I contains the enzymatic activity of NADH dehydrogenase (NDH), and can be isolated as a three subunit subcomplex from submitochondrial particles (SMP) (13). Therefore, the Fp-containing three subunit subcomplex is normally termed as NDH. Of the three subunits, the 51-kDa subunit is an NADH-binding polypeptide hosting one FMN and one [4Fe-4S] iron-sulfur center (N3 center) with an E m,7 ϳ Ϫ250 mV. The 24-kDa subunit hosts one [2Fe-2S] iron-sulfur center (N1a) with an E m,7 ϳ Ϫ370 mV (14, 23). The 9-kDa subunit does not contain any cofactor. NDH retains the same kinetic parameters for NADH dehydrogenation, NADH bin...

. . 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.

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O 2 ·-) and O 2 ·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific Sglutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C 206 and C 187 ) of the 51 kDa subunit and one specific cysteine (C 367 ) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q 1 was significantly enhanced. However, O 2 ·-generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of Sglutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C 206 , C 187 , and C 425 were glutathiolated. C 425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool.Mitochondrial Complex I (EC 1.6.5.3. NADH:ubiquinone oxidoreductase) is the first energyconserving segment of the electron transport chain (ETC) (1-3). The enzyme catalyzes electron transfer from NADH to ubiquinone coupled with the translocation of four protons across the

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.

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