Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System

Background: A device of QCM can be used in the transient kinetics of oxidation of a pair of cysteine residues in DsbA by DsbB. Results: The obtained kinetic parameters indicate that the two pairs of cysteine residues in DsbB are important. Conclusion: The reaction pathway of almost all DsbA oxidation processes would proceed through the stable intermediate. Significance: The transient kinetics of the reaction intermediate is important. Disulfide bond formation protein B (DsbB(S-S,S-S)) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbA(SH,SH)). The oxidized DsbA(S-S), with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbA(SH,SH) and DsbB(S-S,S-S). We immobilized DsbB(S-S,S-S) embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (k(f), k(r), and k(cat), respectively) indicated that the two pairs of cysteine residues in DsbB(S-S,S-S) were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbB(S-S,S-S). Our data suggested that the reaction pathway of almost all DsbA(SH,SH) oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.