Reactivities of quinone-free DsbB from Escherichia coli.
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.
Inaba
Kenji
K
Institute for Virus Research, Kyoto University, and CREST of Japan Science and Technology Agency, Kyoto 606-8507, Japan.
Takahashi
Yoh-Hei
YH
Ito
Koreaki
K
D001426:Bacterial Proteins / Q000502:physiology*; D016227:Benzoquinones / Q000737:chemistry / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012521:Sarcosine / Q000737:chemistry; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D024482:Vitamin K 2 / Q000737:chemistry
D001426:Bacterial Proteins; D016227:Benzoquinones; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D016877:Oxidants; D024482:Vitamin K 2; C004532:quinone; D014364:Tryptophan; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol; D012521:Sarcosine