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

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5-Phosphoribosyl 1-pyrophosphate synthetase (PRibPP synthetase EC 2.7.6.1) isolated from rat intestinal mucosa was found to be membrane associated. The subcellular distribution of PRibPP synthetase activity seems to parallel that of gamma-glutamyl transpeptidase, indicating it to be in the brush border. The tip cells of rat intestinal mucosa were richer in PRibPP synthetase than the crypt cells. Chromatography of a Triton-solubilized particulate fraction unmasked a peak of hypoxanthine phosphoribosyltransferase activity that was not detectable before. The activity, too, was concentrated in the brush border. The coexistence of these two activities in the fraction of the bowl involved in absorption has led to the suggestin that the synthetase and phosphoribosyl-transferase are part of a coupled transport system.