PROTON SHARING BETWEEN CYSTEINE THIOLS IN ESCHERICHIA-COLI THIOREDOXIN - IMPLICATIONS FOR THE MECHANISM OF PROTEIN DISULFIDE REDUCTION

Proton sharing between acidic groups has been observed in the active s ires of several enzymes, including bacteriorhodopsin, aspartic proteas es, and ribonuclease HI. We here report NMR observations suggestive of proton sharing between cysteine thiols in the active site of the oxid ation-reduction enzyme thioredoxin. The pK(a)s of the two cysteine thi ols in the Escherichia coli protein are removed from the expected valu e of 8.4 by similar to 1. pH unit in either direction, upward and down ward. Further, the C beta resonances of both residues show clearly the effects of both of these pK(a)s, indicating that the titrations of th e two thiol groups are intimately linked. This behavior strongly sugge sts that the low pK(a) ascribed to the deprotonation of the Cys 32 thi ol most likely arises through the interaction and close approach of th e thiol of Cys 35, with the thiolate anion of Cys 32 stabilized throug h the sharing of the remaining thiol proton, nominally attached to Cys 35. These observations provide a rationale for the mediation of activ e site pH control, an important aspect of the mechanism of thioredoxin and other proteins with catalytic thioredoxin domains, such as protei n disulfide isomerases.