摘要 : Our group has previously observed that protein S-glutathionylation serves as an integral feedback inhibitor for the production of superoxide (O-2(center dot-))/hydrogen peroxide (H2O2) by alpha-ketoglutarate dehydrogenase (KGDH), ... 展开 Our group has previously observed that protein S-glutathionylation serves as an integral feedback inhibitor for the production of superoxide (O-2(center dot-))/hydrogen peroxide (H2O2) by alpha-ketoglutarate dehydrogenase (KGDH), py-ruvate dehydrogenase (PDH), and complex I in muscle and liver mitochondria, respectively. In the present study, we hypothesized that glutathionylation would fulfill a similar role for the O-2(center dot-)/H2O2 sources sn-glycerol-3-phosphate dehydrogenase (G3PDH), proline dehydrogenase (PRODH), and branched chain keto acid dehydro-genase (BCKDH). Surprisingly, we found that inducing glutathionylation with disulfiram increased the pro-duction of O-2(center dot-)/H2O2 by mitochondria oxidizing glycerol-3-phosphate (G3P), proline (Pro), or alpha-keto-13-methylvaleric acid (KMV). Treatment of mitochondria oxidizing G3P or Pro with rotenone or myxothiazol increased the rate of ROS production after incubating in 1000 nM disulfiram. Incubating mitochondria treated with disulfiram in both rotenone and myxothiazol prevented this increase in O-2(center dot-)/H2O2 production. In addition, when adminstered together, ROS production decreased below control levels. Disulfiram-treated mitochondria displayed higher rates of ROS production when oxidizing succinate, which was inhibited by rotenone, myx-othiazol, and malonate, respectively. Disulfiram also increased ROS production by mitocondria oxidizing KMV. Treatment of mitochondria oxidizing KMV with disulfiram and rotenone or myxothiazol did not alter the rate O-2(center dot-)/H2O2 production further when compared to mitochondria treated with disulfiram only. Analysis of BCKDH activity following disulfiram treatment revealed that glutathionylation does not inhibit the enzyme complex, indicating this alpha-keto acid dehydrogenase is not a target for glutathione modification. However, treatment of mitochondria with rotenone and myxothiazol without disulfiram also augmented ROS production. Overall, we were able to demonstrate for the first time that glutathionylation augments ROS production by the respiratory chain during forward electron transfer (FET) and reverse electron transfer (RET) from the UQ pool. Additionally, we were able to show that BCKDH is not a target for glutathione modification and that glutathionylation can also increase ROS production in mitochondria oxidizing branched chain amino acids following the modification of enzymes upstream of BCKDH. 收起