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 kcat 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.evolution ͉ thiols ͉ disulfide ͉ selenium ͉ enzyme structure

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

“…After the addition of the prosthetic group, the yellow color of the holoenzymes remained stable, indicating interaction of FAD with the protein. Absorption ratios A 280 nm / A 463 nm as well as extinction coefficients of the oxidized enzyme species at 463 nm were determined from spectral analyses (wild type hTrxR: e = 11.3 mM À1 cm À1 [2,3], hTrxR-16 K29R : e = 11.74 mM À1 cm À1 , hTrxRD16 K29R,H108Y : e = 13.87 mM À1 cm À1 , hTrxRD16 K29R,H108Y,A119N,V478E : e = 15.22 mM À1 cm À1 ). Oxidised hTrxR-16 had a typical flavoprotein spectrum [28] with peak absorption at 463 nm ( Fig.…”

“…b 4% of k cat for GSSG reduction (as mentioned for maximal velocity in Ref. [2]); nd, not determined. Fig.…”

“…TrxR catalyzes the NADPH-dependent reduction of Trx disulfide (Trx(S) 2 ). This reaction is comprised of several steps including the transfer of electrons from NADPH to FAD, from FAD to the N-terminally located active site disulfide and subsequently to a second redox center located on the flexible C-terminal end of the other subunit.…”

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“…These enzymes are highly homologous to glutathinone reductase, rather than the small TrxRs, and the N-terminal active sites of large TrxRs are identical to that of glutathione reductase and use a CxxxxC motif in electron transfer to a C-terminal -Gly-Cys-Sec-Gly-active site without conformational changes (353,354). The small and large TrxRs use different mechanisms in catalyzing the reduction of thioredoxin (Trx), which has a conserved Cys-Gly-Pro-Cys active site in all organisms (141,164,272).…”

“…They act both by controlling the function of the central redox molecule thioredoxin, and by directly reducing numerous substrates. TrxRs contain an FAD domain, an NADPH-binding domain, an interphase domain, and a penultimate Sec residue in a 16-residue C-terminal extension, which is indispensable for their enzymatic activity (120,216,353,354). Three mammalian TrxR selenoenzymes have been identified: the cytosolic enzyme TrxR1 (301), the mitochondrial enzyme TrxR2 (197,228), and a testis-specific enzyme thioredoxin-glutathione reductase (TGR/TrxR3), the latter also possessing glutathione and glutaredoxin reductase activity (295,296).…”