Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, h as a marked similarity to glutathione reductase. The two cysteines in the N -terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are con served between them at corresponding positions, but the mammalian thioredox in reductase contains a C-terminal extension of selenocysteine (Sec or U) a t the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly ). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well a s the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin r eductase produced in this system, designated as WT, was found to lack the S ec residue and to terminate at Cys497, A Sec-containing thioredoxin reducta se, which was purified from COS-1 cells transfected with the wild-type cDNA , was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at abo ut three orders magnitude lower than SecWT, WT, U498C, and U498S showed som e 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenas e activity, and C59S and C64S had substantially no such activities. These d ata and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary accepters f or electrons from NADPH via FAD, and that the electrons are then transferre d to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredo xin in the mammalian thioredoxin reductase.