The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of E. coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced state. We split DsbD into three structural domains, each containing two essential cysteines. Remarkably, when coexpressed, these truncated proteins restore DsbD function. Utilizing this three piece system, we were able to determine a pathway of the electrons through DsbD. Our findings strongly suggest that the pathway is based on a series of multistep redox reactions that include direct interactions between thioredoxin and DsbD, and between DsbD and its periplasmic substrates. A thioredoxin-fold domain in DsbD appears to have the novel role of intramolecular electron shuttle.
