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.

Figure 1

Tryptic peptide map of the alkylated oxidized form of bovine TrxR. Solid line indicates absorbance at 214 nm; dashed line, the percent CH₃CN in 0.1% TFA. The S-S bridge and Se-S bridge peptides are labeled P-a and P-c, respectively. The positions of the 13 Cys residues and the SeCys in bovine TrxR are shown (Upper), with peptides identified by sequencing indicated by white boxes and peptides not found in this digest in gray boxes.

Figure 2

Tryptic peptide map of DTT-reduced and double alkylated bovine TrxR. Elution of peptides was monitored by absorbance at 214 nm, 280 nm, and 254 nm [for S-(4-pyridylethyl)-l-cysteine], as shown with solid lines. The dashed line shows the cpm from [¹⁴C]carboxymethylcysteine in each fraction.

Figure 3

Electrospray ionization (ESI) mass spectrum of the C-terminal tryptic peptide from oxidized bovine TrxR. (A Inset) shows the detailed mass spectrum of the peptide detected by ESI, in comparison to the simulated spectra of the peptide with (B) or without (C) a selenenylsulfide.

Figure 4

Sodium dithionite titration of rat SeCys⁴⁹⁸-Cys TrxR. Enzyme (21.2 nmol of subunits) in 0.6 ml of 0.1 M phosphate buffer/1 mM EDTA/pH 7.5 was titrated with 10 mM sodium dithionite dissolved in 50 mM sodium pyrophosphate buffer, pH 8.5, at 25°C in the presence of 150 nM methyl viologen. Spectra show oxidized enzyme (curve 1), the enzyme reduced with 0.94 equivalents (curve 2), 1.4 equivalents (curve 3), 1.9 equivalents (curve 4), and 2.83 equivalents of dithionite (curve 5). A slight artifact in the spectra at 360 nm is because of lamp change at this wavelength. Inset shows the effects of dithionite addition on the absorbances at 464 nm and at 540 nm.

Figure 5

Proposed model of mammalian TrxR based on homology to GR. (Upper) Model of GR with FAD, NADPH, and interface domains (adapted from refs. 1, 6, 13, 14). Glutathione disulfide bound is indicated as well as the active-site cysteines (black circles). (Lower) Model of mammalian TrxR. The elongation with 16-residues is extending from the interface, positioning SeCys⁴⁹⁸ and Cys⁴⁹⁷ in one subunit adjacent to Cys⁵⁹ and Cys⁶⁴ of the other subunit.

Figure 6

Simplified postulated mechanism for hydrogen peroxide reduction by mammalian TrxR. See Discussion.

Figure 7

Postulated mechanism for Trx reduction by mammalian TrxR. See Discussion.