Conditions Conducive to the Glutathionylation of Complex I Subunit NDUFS1 Augment ROS Production following the Oxidation of Ubiquinone Linked Substrates, Glycerol-3-Phosphate and Proline.

Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O2●-/H2O2) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O2●-/H2O2 production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50-1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies.

The Uncoupling Proteins: A Systematic Review on the Mechanism Used in the Prevention of Oxidative Stress.

Mitochondrial uncoupling proteins (UCP) 1-3 fulfill many physiological functions, ranging from non-shivering thermogenesis (UCP1) to glucose-stimulated insulin release (GSIS) and satiety signaling (UCP2) and muscle fuel metabolism (UCP3). Several studies have suggested that UCPs mediate these functions by facilitating proton return to the matrix. This would decrease protonic backpressure on the respiratory chain, lowering the production of hydrogen peroxide (H2O2), a second messenger. However, controlling mitochondrial H2O2 production to prevent oxidative stress by activating these leaks through these proteins is still enthusiastically debated. This is due to compelling evidence that UCP2/3 fulfill other function(s) and the inability to reproduce findings that UCP1-3 use inducible leaks to control reactive oxygen species (ROS) production. Further, other studies have found that UCP2/3 may serve as Ca2+. Therefore, we performed a systematic review aiming to summarize the results collected on the topic. A literature search using a list of curated keywords in Pubmed, BIOSIS Citation Index and Scopus was conducted. Potentially relevant references were screened, duplicate references eliminated, and then literature titles and abstracts were evaluated using Rayyan software. A total of 1101 eligible studies were identified for the review. From this total, 416 studies were evaluated based on our inclusion criteria. In general, most studies identified a role for UCPs in preventing oxidative stress, and in some cases, this may be related to the induction of leaks and lowering protonic backpressure on the respiratory chain. However, some studies also generated evidence that UCP2/3 may mitigate oxidative stress by transporting Ca2+ into the matrix, exporting lipid hydroperoxides, or by transporting C-4 metabolites. Additionally, some showed that activating UCP1 or 3 can increase mitochondrial ROS production, even though there is still augmented protection from oxidative stress. Conclusion: Overall, most available studies demonstrate that UCPs, particularly UCP2/3, prevent oxidative stress. However, the mechanism utilized to do so remains elusive and raises the question that UCP2/3 should be renamed, since they may still not be true "uncoupling proteins".

The glutathionylation agent disulfiram augments superoxide/hydrogen peroxide production when liver mitochondria are oxidizing ubiquinone pool-linked and branched chain amino acid substrates.

Our group has previously observed that protein S-glutathionylation serves as an integral feedback inhibitor for the production of superoxide (O2●-)/hydrogen peroxide (H2O2) by α-ketoglutarate dehydrogenase (KGDH), pyruvate 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 O2●-/H2O2 sources sn-glycerol-3-phosphate dehydrogenase (G3PDH), proline dehydrogenase (PRODH), and branched chain keto acid dehydrogenase (BCKDH). Surprisingly, we found that inducing glutathionylation with disulfiram increased the production of O2●-/H2O2 by mitochondria oxidizing glycerol-3-phosphate (G3P), proline (Pro), or α-keto-ß-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 O2●-/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, myxothiazol, 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 O2●-/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 α-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.