The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Qing Cheng,William E Antholine,Elias S.J Arnér,Judith M Myers,Balaraman Kalyanaraman,Charles R Myers

doi:10.1074/jbc.m110.117259

Qing Cheng, William E Antholine + Show 4 more

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https://doi.org/10.1074/jbc.m110.117259

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Abstract

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.

Highlights

Mammalian thioredoxin reductase (TrxR)4 is a ubiquitous NADPH-dependent flavoenzyme that has prominent disulfide reductase activity
The major forms of TrxRs are NADPH-dependent homodimers, and each subunit contains three redox-active sites [1, 3, 4, 7, 8]: (a) one FAD that accepts electrons from NADPH, (b) an N-terminal domain dithiol (-CVNVGC- whose Cys residues occupy positions Cys59/Cys64 in TrxR1 studied here) that is reduced by the flavin, and (c) the C-terminal active site, which is believed to accept electrons from the N-terminal dithiol and contains selenocysteine (Sec) within the sequence -Gly-Cys-Sec-Gly [8]
Assay for TrxR Activity—The activity of purified TrxR was measured as the NADPH-dependent reduction of 5,5Ј-dithiobis(2-nitrobenzoic) acid (DTNB) [20, 34]

Summary

Introduction

Mammalian thioredoxin reductase (TrxR) is a ubiquitous NADPH-dependent flavoenzyme that has prominent disulfide reductase activity. The electron flow of the natural catalytic cycle of mammalian TrxR (i.e. catalyzing the reduction of substrates, such as oxidized Trx or lipoic acid) may be diverted to O2 under certain conditions, converting the enzyme to a prooxidant NADPH oxidase. This was first described for the TrxRmediated reduction of selenite to selenide, which, when reoxidized to selenite, can mimic a strong NADPH oxidase activity through oxygen-coupled redox cycling [5]. The catalytic mechanisms of the non-induced inherent NADPH oxidase activity of TrxR have not yet been elucidated

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