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Dihydroorotate dehydrogenase (DHODH) oxidizes dihydroorotate to orotate for pyrimidine biosynthesis, donating electrons to the ubiquinone (UQ) pool of mitochondria. DHODH has a measurable rate for hydrogen peroxide (H

Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O2•-/ H2O2 emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O2•-/ H2O2 production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD+ lowering O2•-/ H2O2 production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O2•-/ H2O2 production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.

Reactive oxygen species (ROS), natural by-products of aerobic respiration, are important cell signaling molecules, which left unchecked can severely impair cellular functions and induce cell death. Hence, cells have developed a series of systems to keep ROS in the nontoxic range. Uncoupling proteins (UCPs) 1-3 are mitochondrial anion carrier proteins that are purported to play important roles in minimizing ROS emission from the electron transport chain. The function of UCP1 in this regard is highly contentious. However, UCPs 2 and 3 are generally thought to be activated by ROS or ROS by-products to induce proton leak, thus providing a negative feedback loop for mitochondrial ROS production. In our laboratory, we have not only confirmed that ROS activate UCP2 and UCP3, but also demonstrated that UCP2 and UCP3 are controlled by covalent modification by glutathione. Furthermore, the reversible glutathionylation is required to activate/inhibit UCP2 and UCP3, but not UCP1. Hence, our findings are consistent with the notion that UCPs 2 and 3 are acutely activated by ROS, which then directly modulate the glutathionylation status of the UCP to decrease ROS emission and participate in cell signaling mechanisms.