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-2(raical anion)), 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 O2(radical anion). adduct (DEPMPO/HOO center dot) to the hydroxyl radical adduct(DEPMPO/HO center dot). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O-2(radical anion). Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys59/Cys64 dithiol is required for O-2(radical anion) generation and that O-2(radical anion) is not derived directly from the FAD. Mutations in the Cys59/Cys64 dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys497/Sec498 active site. Although the bulk of the DEPMPO/HO center dot 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 center dot could be generated by the enzyme in an H2O2-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.