Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions.
Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress. However, its exact antioxidant nature has not been elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study. In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM. Both E. coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry. The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin. Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties. In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin. Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin.
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Department of Molecular Biology, University of Texas Health Center at Tyler, 11937 U.S. Highway 271, Tyler, Texas 75708, USA. kumuda@uthct.edu
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D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002384:Catalysis; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004234:Diuretics, Osmotic / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry; D016166:Free Radical Scavengers / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism*; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008353:Mannitol / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013481:Superoxides; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013890:Thiourea / Q000494:pharmacology; D013997:Time Factors
D001426:Bacterial Proteins; D003497:Cyclic N-Oxides; D004234:Diuretics, Osmotic; D016166:Free Radical Scavengers; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D013481:Superoxides; D017665:Hydroxyl Radical; D008353:Mannitol; D013879:Thioredoxins; D013890:Thiourea; D003545:Cysteine; D000409:Alanine; D010100:Oxygen; C003959:TEMPO