The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.
Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.
Cheng
Qing
Q
Department of Pharmacology and Toxicology, MedicalCollege of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Antholine
William E
WE
Myers
Judith M
JM
Kalyanaraman
Balaraman
B
Arnér
Elias S J
ES
Myers
Charles R
CR
D000818:Animals; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009154:Mutation; D019255:NADPH Oxidases / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012643:Selenium / Q000737:chemistry*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry
C510912:(diethoxyphosphoryl)-5-methylpyrroline N-oxide; D016877:Oxidants; D011758:Pyrroles; D011994:Recombinant Proteins; D017665:Hydroxyl Radical; D013879:Thioredoxins; D009195:Peroxidase; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium
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