Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulp hide bridges on target proteins, The catalytic mechanism proceeds via a mix ed disulphide intermediate whose breakdown should be enhanced by the involv ement of a conserved buried residue, Asp-30, as a base catalyst towards res idue Cys-39. We report here the crystal structure of wild-type and D30A mut ant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the fir st crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer , A careful analysis of all available crystal structures reveals that the r elative positioning of residues Asp-30 and Cys39 as well as hydrophobic con tacts in the vicinity of residue Asp-30 do not allow a conformational chang e sufficient to bring the two residues close enough for a direct proton tra nsfer. This suggests that protonation/deprotonation of Cys-39 should be med iated by a water molecule. Molecular-dynamics simulations, carried out eith er in vacuo or in water. as well as proton-inventory experiments, support t his hypothesis. The results are discussed with respect to biochemical and s tructural data.