Abstract
DsbB is a disulfide oxidoreductase present in the Escherichia coli plasma membrane. Its cysteine pairs, Cys41-Cys44 and Cys104-Cys130, facing the periplasm, as well as the bound quinone molecules play crucial roles in oxidizing DsbA, the protein dithiol oxidant in the periplasm. In this study, we characterized quinone-free forms of DsbB prepared from mutant cells unable to synthesize ubiquinone and menaquinone. While such preparations lacked detectable quinones, previously reported lauroylsarcosine treatment was ineffective in removing DsbB-associated quinones. Moreover, DsbB-bound quinone was shown to contribute to the redox-dependent fluorescence changes observed with DsbB. Now we reconfirmed that redox potentials of cysteine pairs of quinone-free DsbB are lower than that of DsbA, as far as determined in dithiothreitol redox buffer. Nevertheless, the quinone-free DsbB was able to oxidize approximately 40% of DsbA in a 1:1 stoichiometric reaction, in which hemi-oxidized forms of DsbB having either disulfide are generated. It was suggested that the DsbB-DsbA system is designed in such a way that specific interaction of the two components enables the thiol-disulfide exchanges in the "forward" direction. In addition, a minor fraction of quinone-free DsbB formed the DsbA-DsbB disulfide complex stably. Our results show that the rapid and the slow pathways of DsbA oxidation can proceed up to significant points, after which these reactions must be completed and recycled by quinones under physiological conditions. We discuss the significance of having such multiple reaction pathways for the DsbB-dependent DsbA oxidation.
Highlights
Anaerobic conditions, respectively [3,4,5,6]
DsbB is integrated into the membrane via its four transmembrane segments, with cytoplasmic termini, a short cytoplasmic loop, and two periplasmic regions
Each of its periplasmic regions contains a pair of cysteines, Cys41–Cys44 and Cys104–Cys130, respectively, which are essential for its function [7]
Summary
Anaerobic conditions, respectively [3,4,5,6]. Whereas it is believed that the respiratory quinones enable the DsbB-DsbA cascade to cycle catalytically, there are conflicting views about the basic redox reactivity of DsbB. Grauschopf et al [13] argued subsequently that DsbB-bound UQ must be removed and direct spectroscopic measurements rather than the chemical modification of free thiols must be used to accurately estimate the redox potentials of the DsbB active sites. They attempted to remove the bound UQ by LS treatment of the DsbB preparation and measured fluorescence changes of the preparation in different redox buffers. Their results showed that Cys41–Cys in DsbB was even more oxidizing (Ϫ69 mV) than Cys30–Cys of DsbA. We discuss how individual elements in this system are integrated into the elaborate multiple mechanisms that ensure effective recycling of the disulfide bond formation reactions, in which quinone molecules play essential roles
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