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.