Crystal structure of the Wild-Type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic machanism

Thioredoxins are ubiquitous proteins which catalyze the redn. of disulfide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulfide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first 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 sulfur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful anal. of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water mol. Mol.-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory expts., support this hypothesis. The results are discussed with respect to biochem. and structural data.