REDUCTION OF PHENOXYL RADICALS BY THIOREDOXIN RESULTS IN SELECTIVE OXIDATION OF ITS SH-GROUPS TO DISULFIDES - AN ANTIOXIDANT FUNCTION OF THIOREDOXIN

Thioredoxin is an important cellular redox buffer. In this report, we describe the reaction of thioredoxin with phenoxyl radicals. The vicin al sulfhydryls of the bis(cysteinyl) active site sequence reduced phen oxyl radicals released in horseradish peroxidase-catalyzed oxidation o f phenol. Redox cycling of phenol was accompanied by selective oxidati on of thioredoxin sulfhydryls to disulfides. HPLC/UV-vis measurements showed that the SH:phenol oxidation ratio was 15:1 under the condition s used. At the end of the reaction, oxidized thioredoxin was quantitat ively recovered in the reduced form with dithiothreitol. Oxidation of sulfhydryls to sulfoxy derivatives, oxidation of other amino acid resi dues, and formation of covalent adducts with phenolic metabolites (qui nones) were not detected by LC-MS, While the thiyl radical of glutathi one was readily detected with the spin trap 5,5-dimethyl-1-pyrroline N -oxide, no ESR-detectable DMPO-thiyl adducts formed during the oxidati on of thioredoxin. Similarly, oxidation of vicinal sulfhydryls of dihy drolipoic acid did not produce DMPO-thiyl spin adducts, indicating tha t fast intramolecular cyclization to disulfide occurred with thioredox in. Measurements of the superoxide dismutase-sensitive chemiluminescen ce response of lucigenin demonstrated that thioredoxin oxidation was a ccompanied by release of superoxide, most likely via disulfide radical anion-mediated one-electron reduction of oxygen. We propose that form ation of disulfides is characteristic of the phenoxyl radical-catalyze d oxidation of vicinal sulfhydryls in both small thiols and disulfide- forming oxidoreductases. Reversibility of the phenoxyl radical-catalyz ed modification of thioredoxin may be responsible for its function as an efficient cytosolic antioxidant.