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The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

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.

SUBMITTER: Cheng Q

PROVIDER: S-EPMC2898413 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

Cheng Qing Q Antholine William E WE Myers Judith M JM Kalyanaraman Balaraman B Arnér Elias S J ES Myers Charles R CR

The Journal of biological chemistry 20100510 28

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 w ...[more]

PMID: 20457604

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Project description:Cytosolic thioredoxin reductase 1 (TR1) is the best characterized of the class of high-molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in a large decrease in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. The Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high-Mr TRs from other organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum, do not require Sec to reduce small molecule substrates, including 5,5'-dithiobis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data show that high-Mr TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum, and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a "guiding bar", amino acids 407-422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and co-workers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. J. Mol. Biol. 370, 116-127], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec → Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes, in contrast, are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater access of the substrate to the N-terminal redox center and because they can make use of alternative mechanistic pathways that are not available to TR1.

Project description:Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.

Project description:Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox-active tetrapeptide of sequence Gly-Cys(1)-Sec(2)-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulysin, HIV Tat protein, H(2)O(2), lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys(2) mutant enzyme or the N-terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild-type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175-550-fold reduction in k(cat). Our data support the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, or (iii) that are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC(50) for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83-142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.

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Dataset Information

The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

Publications

The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

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