Reconstitution of a protein disulfide catalytic system

Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a Km = 10 microM. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.