Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: Redox independent functions

Thioredoxin is a ubiquitous small protein known to protect cells and tissue s against oxidative stress. However, its exact antioxidant nature has not b een elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 muM caused 50% inhibition of TEMP-O-1(2) (TEMPO) adduct f ormation in a photolysis EPR study. In contrast, Escherichia coli thioredox in caused 50% inhibition of TEMPO formation at 80 muM. Bath E. coli thiored oxin and human thioredoxin inhibited . OH dependent DMPO-OH formation. as d emonstrated by EPR spectrometry, The quenching of O-1(2) or scavenging of . OH was not dependent upon the redox state of thioredoxin. Using a human th ioredoxin in which the structural cysteines were mutated to alanine, Trx-C3 A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scaven ging properties. In addition, using a quadruple mutant Trx-C4A, where one o f the catalytic cysteines, C35 was mutated to alanine in addition to the mu tated structural cysteines, we demonstrated that catalytic cysteines are al so required for the scavenging action of thioredoxin. Identification of thi oredoxin as a O-1(2) quencher and . OH scavenger may be of significant impo rtance in explaining various redox-related antioxidant functions of thiored oxin. (C) 2000 Academic Press.