REDUCTION OF THE ASCORBYL FREE-RADICAL TO ASCORBATE BY THIOREDOXIN REDUCTASE

Recycling of ascorbic acid from its oxidized forms is required to main tain intracellular stores of the vitamin in most cells. Since the ubiq uitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorb ate, Purified rat liver thioredoxin reductase catalyzed the disappeara nce of NADPH in the presence of low micromolar concentrations of the a scorbyl free radical that were generated from ascorbate by ascorbate o xidase, and this effect was markedly stimulated by selenocystine, Dehy droascorbic acid is generated by dismutation of the ascorbyl free radi cal, and thioredoxin reductase can reduce dehydroascorbic acid to asco rbate. However, control studies showed that the amounts of dehydroasco rbic acid generated under the assay conditions used were too low to ac count for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-st ate ascorbyl free radical concentrations, as expected if thioredoxin r eductase reduces the ascorbyl free radical, Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the asco rbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selen ium-deficient rats, by inhibition with aurothioglucose at concentratio ns selective for thioredoxin reductase, and by stimulation with seleno cystine. Microsomal fractions prepared from rat liver showed substanti al NADH-dependent ascorbyl free radical reduction that was not sensiti ve to selenium depletion. These results suggest that thioredoxin reduc tase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dep endent reductases.