A MIXED SELENODISULPHIDE BOND AT THE ACTIVE SITE OF THIOREDOXIN REDUCTASE

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Mammalian cytosolic thioredoxin reductase (TrxR) has a redox-sensitive Cys59 and Cys64 pair adjacent to the flavin ring and a Cys497 and Secys498 pair near the COOH-terminus. To characterize the chemical nature of the Secys residue in oxidized TrxR, TrxR was treated with H2O2 or oxidized thioredoxin.. The resulting enzymes were then labeled with biotin-conjugated iodoacetamide followed by iodoacetamide and cleaved with Lys-C, and finally, the digested peptide mixtures were analyzed by LC/MS. Total ion chromatograms included peaks with a mass of 1297.7 daltons (retention time, 20.5 min) which is essentially identical, respectively, to the 1297.3 daltons expected for the COOH- terminal fragment R487SGGDILQSGCUG499 containing Cys497 and Secys498 (denoted by one-letter codon U) bridged by a mixed selenodisulfide bond (S-Se peptide). Edman degradation of the 20.5-min peptide yielded a complete sequence of RSGGDILQSG. Together with the mass data, the sequencing result conclusively proves that the 20.5-min peptide is indeed the S-Se peptide. In the presence of limited amounts of TrxS2, the active site Cys-SH/Cys-SH pair was preferentially oxidized over the Cys-SH/Secys-SeH pair. Both the oxidation of the Cys-SH/Cys-SH pair by TrxS2 or H2O2 and the reduction of the Cys/Cys disulfide by NADPH were inhibited by alkylation of the Secys residue. Furthermore, the Secys modification completely blocked the ability of TrxR to catalyze the reduction of TrxS2 in the presence of NADPH. These results suggest that the function of the Cys/Secys pair may be essential to pass reducing equivalents from the active site Cys-SH/Cys-SH pair to TrxS2 . - thioredoxin; thioredoxin reductase; slenium, selenosulphide bond