Characterization of the menaquinone-dependent disulfide bond formation pathway of Escherichia coli.
In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys41 and Cys44 of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking >1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB(MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.
Takahashi
Yoh-hei
YH
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Inaba
Kenji
K
Ito
Koreaki
K
D000933:Antifibrinolytic Agents / Q000494:pharmacology*; D001667:Binding, Competitive; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011809:Quinones / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors; D024482:Vitamin K 2 / Q000494:pharmacology*
D000933:Antifibrinolytic Agents; D004220:Disulfides; D011809:Quinones; D024482:Vitamin K 2; D003545:Cysteine; D010100:Oxygen