How Thioredoxin Dissociates Its Mixed Disulfide

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29 Trx -Cys89 ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29 Trx on the exposed Cys82 ArsC -Cys89 ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32 Trx in contact with Cys29 Trx . Cys32 Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32 Trx is found to be more reactive than Cys82 ArsC . Additionally, Cys32 Trx directs its nucleophilic attack on the more susceptible Cys29 Trx and not on Cys89 ArsC . This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.