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Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence.

ABSTRACT: Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys(59)-Val-Asn-Val-Gly-Cys(64), identical to the active site of GR; the other was a selenenylsulfide formed from Cys(497)-SeCys(498) and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys(497)-SeCys(498) residues of one monomer are adjacent to the Cys(59) and Cys(64) residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys(59) and Cys(64) dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys(498) replaced by Cys having a 100-fold lower k(cat) for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys(59)-Cys(64) was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.

SUBMITTER: Zhong L

PROVIDER: S-EPMC18523 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence.

Zhong L L Arnér E S ES Holmgren A A

Proceedings of the National Academy of Sciences of the United States of America 20000501 11

Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys(59)-Val-Asn-Val-Gly-Cys(64), identical to the active site of GR; the other was a selenenylsulfide formed from ...[more]

PMID: 10801974

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Project description:Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1-4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

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.

Dataset Information

Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence.

Publications

Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence.

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