REDOX POTENTIALS OF ACTIVE-SITE BIS(CYSTEINYL) FRAGMENTS OF THIOL-PROTEIN OXIDOREDUCTASES

The active sites of thiol-protein oxidoreductases consist of the chara cteristic Cys-X-X-CYS motif, and the redox potentials of these enzymes reflect the propensity of the bis(cysteinyl) sequence portion for dis ulfide loop formation. Thereby, as is known from comparing the three-d imensional (3D) structures of thioredoxin and glutaredoxin in the redu ced and oxidized state, reduction of the disulfide bond is accompanied by minimal perturbation of the backbone folding of the active sites. In order to estimate the sequence-dependent intrinsic free energy of f ormation of the active-site disulfide loops in oxidoreductases, synthe tic fragments corresponding to the sequences 31-38, 10-17, 134-141, an d 34-41 of thioredoxin, glutaredoxin, thioredoxin reductase, and prote in disulfide isomerase (PDI), respectively, were analyzed for their te ndency to form 14-membered rings. For this purpose thiol/disulfide exc hange experiments, with glutathione as reference redox pair, were perf ormed on the bis(cysteinyl) octapeptides. As the free energy of ring c losure of linear peptides consists mainly of the free energy of format ion of the disulfide loop with a defined geometry from a statistical e nsemble of conformations of the bis(cysteinyl) peptides, the observed differences in the equilibrium constants, although relatively small (w ithin a factor 10), suggest that sequence-dependent information for lo op formation is retained in the excised active-site fragments. These i nherent redox potentials are, however, significantly affected and/or a mplified in the native proteins by the conformational restraints impos ed by the ''structural domains'' on the ''functional domains''.