Disulfide bond formation involves a quinhydrone-type charge–transfer complex

Journal of Biological Chemistry

2004

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.

Section: Discussionsupporting

confidence: 53%

mentioning

confidence: 99%

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

Journal of Biological Chemistry

2004

“…Finally, we point out that at the final re-revision stage of this article, we came across an internet pre-release of a publication from Bardwell and coworkers (30). These authors described that wild-type DsbB is purple-colored and attempted to explain this observation in terms of the purple-colored quinhydrone, in which two stacked quinone molecules are involved.…”

Section: Uq Is Red-shifted Transiently During Reaction Betweenmentioning

confidence: 96%

“…Bacterium Escherichia coli has a series of disulfide bond formation factors (Dsb) 1 in the periplasmic space-plasma membrane region of the cell (1)(2)(3). DsbA is a periplasmic enzyme having a thioredoxin-like fold and is capable of donating its Cys 30 -Cys 33 disulfide to newly translocated substrate proteins (4 -7). Then, the two active site cysteines must be reoxidized for the continuing catalysis, and a plasma membrane protein, DsbB, is responsible for this reoxidation (8 -10).…”

mentioning

confidence: 99%

DsbB Elicits a Red-shift of Bound Ubiquinone during the Catalysis of DsbA Oxidation

Journal of Biological Chemistry

2004

102

DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys 30 -Pro-His-Cys 33 active site of DsbA, the primary dithiol oxidant in the periplasm. Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule. This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color. The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB. Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood of Cys 44 . We propose that the Cys 44 -induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.Oxidative folding is crucial for the maturation of extracytoplasmic domains of membrane and secreted proteins. Bacterium Escherichia coli has a series of disulfide bond formation factors (Dsb) 1 in the periplasmic space-plasma membrane region of the cell (1-3). DsbA is a periplasmic enzyme having a thioredoxin-like fold and is capable of donating its Cys 30 -Cys 33 disulfide to newly translocated substrate proteins (4 -7). Then, the two active site cysteines must be reoxidized for the continuing catalysis, and a plasma membrane protein, DsbB, is responsible for this reoxidation (8 -10). In vivo studies showed that the respiratory components of the cell are required for the maintenance of the oxidized state of DsbB, thus providing an oxidizing equivalent to the DsbA/DsbB system (11,12). In vitro studies showed that UQ directly activates DsbB for the DsbAoxidizing activity (13,14 UQ (12,19,20). However, recent studies suggest that the above-mentioned serial arrangement is too simple to account for the actual molecular mechanism of the DsbB-mediated DsbA oxidation reaction. Several questions have been asked. First, are the sequence of reactions driven simply by the intrinsic redox potential differences of the cysteine pairs involved? The experimental results of Inaba and Ito (20) as well as those of Regeimbal and Bardwell (21) gave a negative answer to this question. Specifically, the redox potential of Cys 104 -Cys 130 in DsbB (Ϫ250 mV) is much lower than that of Cys 30 -Cys 33 in DsbA (Ϫ120 mV) such that their in vitro reactions proceed only in the "reverse" direction. The above authors also showed that redox potential of the Cys 41 -Cys 44 pair is again lower (Ϫ210 to Ϫ270 mV) than that of DsbA. More recently, however, Grauschopf et al. (22) reached the opposite conclusion for the Cys 41 -Cys 44 redox potential (estimated to be Ϫ70 mV), using DsbB preparations chemically deprived of UQ. The second question is whether or not the reaction proceeds by sequential rearrangements of inter-and intramolecular disulf...

“…Regeimbal et al (17) reported that their DsbB preparation was purple-colored and discussed that it was due to the formation of quinhydrone-like charge transfer (CT) complex between UQ and hydroquinone that was also bound to DsbB. However, the lack of supporting evidence other than the similarity of the absorbance curve with that of quinhydrone made us question the likelihood of the involvement of the stacked two quinone rings for the spectroscopic behavior of DsbB.…”

mentioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created de novo. DsbB, an Escherichia coli plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink ( max, Ϸ500 nm, ubiquinone) or violet ( max, Ϸ550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the ␣-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanidinium moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at Ϸ500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adduct that accepts a nucleophilic attack from another cysteine to generate a disulfide bond de novo. A similar mechanism is conceivable for a class of eukaryotic dithiol oxidases having a FAD cofactor.FAD ͉ ubiquinone ͉ DsbA