Characterization of the Menaquinone-dependent Disulfide Bond Formation Pathway of Escherichia coli

Abstract

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 Cys 41 and Cys 44 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 ( max ‫؍‬ 550 nm), which required a reduced state of Cys 44 as shown previously for UQ color development ( max ‫؍‬ 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30°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 DsbADsbB redox reactions.

Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB

“…Indeed, a series of our mutational analyses indicated that the quinone transition takes place whenever Cys-44 is unpaired. Consistent with the involvement of Cys-44, the transition is sensitive to a thiol modification agent and to pH changes, giving an apparent pK a of Ϸ6.6 expected for thiolate deprotonation (6,16).…”

“…We have characterized reactivities and properties of DsbB that is free from any bound quinones (11), of DsbB that bears UQ (16), and of DsbB that bears menaquinone (6). These analyses taken together revealed that there are two pathways of DsbB͞ quinone-mediated oxidation of DsbA.…”

“…DsbB is associated with a respiration-coupled cofactor, either aerobic ubiquinone (UQ8) or anaerobic menaquinone (MK8) (4)(5)(6). Each of its two periplasmic domains carries a pair of essential cysteine residues, Cys-41-Cys-44 and Cys-104-Cys-130, respectively (7).…”

Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase

“…Bekker et al (6) reported significant changes in both size and redox state of the Q pool when the environment changes from a well aerated environment to an environment with low oxygen availability. Some respiratory components that intrinsically interact with Q are capable of catalyzing redox reactions with both UQ and MQ (7)(8)(9)(10)(11). Such interactions may be important in physiological and evolutionary aspects in addition to enzymatic function.…”

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…Arg48 substitutions in DsbB result in a low-activity enzyme that can no longer utilize the MK analog menadione as an in vitro electron-accepting substrate (Kadokura et al, 2000). MK-8 has been shown to associate with DsbB, similar to that of UQ, by spectroscopic analysis (Takahashi et al, 2004). The in vitro reaction of DsbA oxidation with MK-8 has been shown to be slower than the UQ-dependent reaction.…”