Our group has found that protein S-glutathionylation serves as an important feedback inhibitor for superoxide (O)/hydrogen peroxide (HO) production by several mitochondrial dehydrogenases. Since cytoplasmic oxidases can also serve as important reactive oxygen species (ROS) sources, we hypothesized that glutathionylation can also inhibit O/HO by these enzymes. We first focused our attention on using a purified xanthine oxidase (XO) of bacterial origin to discern if glutathionylation can shut down ROS production by this enzyme. Incubating XO in glutathione disulfide (GSSG) at a final concentration of 1 mM did not significantly alter ROS production. Additionally, incubating samples in up to 10 mM GSSG increased ROS production. However, diamide and disulfiram titrations in the presence of 1 mM GSH revealed that both glutathionylation catalysts were able to abolish O/HO by XO. Exposure of XO to glutaredoxin-1 (GRX1) and GSSG did not alter the rate of O/HO production. However, incubation with GSH and purified glutathione S-transferase (GST) almost abolished ROS production by XO. Similar results were collected with rat liver cytoplasm. Indeed, diamide and disulfiram significantly decreased ROS production by xanthine oxidoreductase (XOR). Additionally, incubating the cytoplasm in GSH and GST led to a significant decrease in XOR activity. Immunoblot analyses revealed that immunoreactive bands corresponding to XOR were glutathionylated by diamide. Collectively, our findings demonstrate for the first time that cytoplasmic ROS sources, such as XOR, can also be inhibited by glutathionylation and these reactions are enzymatically mediated by GST. Additionally, we found that bacterial XO is also a target for glutathionylation.
