The functional role of selenocysteine (Sec) in the catalysis mechanism of large thioredoxin reductases: proposition of a swapping catalytic triad including a Sec-His-Glu state.

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X-ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol(-1), it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol(-1) more energy, which corresponds to a calculated equilibrium shift of approximately 1000:1 or a 10(3) rate acceleration: a value close to the experimental one of about 10(2) times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.