title: Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity.
abstract: Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys(59)/Cys(64) adjacent to the flavin ring of FAD and another center consisting of Cys(497)/selenocysteine (SeCys)(498) near the C terminus. We now show that the C-terminal Cys(497)-SH/SeCys(498)-Se(-) of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS(2)) or H(2)O(2). The Cys(59)-SH/Cys(64)-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS(2), the Cys(59)-SH/Cys(64)-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys(497)-SH/SeCys(498)-Se(-). Specific alkylation of SeCys(498) completely blocked the TrxS(2)-induced oxidation of Cys(59)-SH/Cys(64)-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys(498) with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys(497)/Cys(498) mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5, 5'-dithiobis(2-nitrobenzoic acid) or TrxS(2), respectively, as substrate. Disulfide formation induced by excess TrxS(2) in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.
authors.affiliation: Laboratories of Cell Signaling and Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Bar-Noy
authors.forename: J
authors.initials: J
authors.lastname: Kwon
authors.forename: R L
authors.initials: RL
authors.lastname: Levine
authors.forename: T C
authors.initials: TC
authors.lastname: Stadtman
authors.forename: S G
authors.initials: SG
authors.lastname: Rhee
mesh_terms: D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology; D011485:Protein Binding; D051381:Rats; D012643:Selenium / Q000737:chemistry*; D017279:Selenocysteine / Q000737:chemistry*; D013053:Spectrophotometry; D013455:Sulfur / Q000737:chemistry*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
chemical_list: D017279:Selenocysteine; D013455:Sulfur; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium; D003545:Cysteine