Kinetic Characterization of the Disulfide Bond-forming Enzyme DsbB

DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at ∼510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-Box and DsbAred, there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k1 ∼5 × 105 m–1 s–1), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k2 ∼ 2 s–1; k3 ∼ 0.3 s–1), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k2 and k3 are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle. DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at ∼510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-Box and DsbAred, there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k1 ∼5 × 105 m–1 s–1), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k2 ∼ 2 s–1; k3 ∼ 0.3 s–1), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k2 and k3 are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle. Disulfide bonds are post-translational modifications that play vital roles in the folding, stability, and activity of numerous secreted proteins. In the Escherichia coli periplasm, disulfide bond formation is catalyzed by the DsbA/DsbB system (for reviews see Refs. 1Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (448) Google Scholar and 2Nakamoto H. Bardwell J.C. Biochim. Biophys. Acta. 2004; 1694: 111-119Crossref PubMed Scopus (151) Google Scholar). DsbA donates its disulfide bond directly to substrate proteins (3Frech C. Wunderlich M. Glockshuber R. Schmid F.X. EMBO J. 1996; 15: 392-398Crossref PubMed Scopus (50) Google Scholar, 4Zapun A. Creighton T.E. Biochemistry. 1994; 33: 5202-5211Crossref PubMed Scopus (88) Google Scholar), and to remain catalytically active, DsbA must be reoxidized by an electron acceptor. This role is filled by the enzyme DsbB, which catalyzes the de novo formation of disulfide bonds using the oxidizing power of a bound ubiquinone cofactor, which in turn links disulfide bond formation to the electron transport chain (5Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 6Bader M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 7Bader M.W. Xie T. Yu C.A. Bardwell J.C. J. Biol. Chem. 2000; 275: 26082-26088Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8Kobayashi T. Ito K. EMBO J. 1999; 18: 1192-1198Crossref PubMed Scopus (92) Google Scholar, 9Kobayashi T. Kishigami S. Sone M. Inokuchi H. Mogi T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11857-11862Crossref PubMed Scopus (208) Google Scholar). Although disulfide exchange reactions have been well studied in many systems, much less is known about how they are formed de novo. Because DsbB is the primary source of disulfides in the cell, it is of great interest to elucidate its catalytic mechanism. In the process of transferring electrons from DsbA to ubiquinone, DsbB undergoes a spectral transition to a purple species (λmax ∼ 510 nm). This was first suggested to be due to the formation of a quinhydrone, a stacked ring system consisting of one oxidized and one reduced quinone molecule (10Regeimbal J. Gleiter S. Trumpower B.L. Yu C.A. Diwakar M. Ballou D.P. Bardwell J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13779-13784Crossref PubMed Scopus (67) Google Scholar). More recent studies support an alternative explanation in which the Cys44 thiolate of DsbB is involved in a charge transfer complex with a DsbB-bound ubiquinone (11Inaba K. Takahashi Y.H. Fujieda N. Kano K. Miyoshi H. Ito K. J. Biol. Chem. 2004; 279: 6761-6768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Inaba K. Takahashi Y.H. Ito K. Hayashi S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 287-292Crossref PubMed Scopus (55) Google Scholar). This was supported by the observation that in a DsbB mutant, which only contains only one cysteine (Cys44), a stable purple color is observed. This purple color is independent of DsbA, but it requires quinone. Although the purple intermediate of DsbB is associated with a free Cys44, it is likely that subsequent steps of the reaction, namely the regeneration of an oxidized ubiquinone on DsbB, involve a quinhydrone-like complex. An intriguing and controversial aspect of the DsbA/DsbB system is the paradoxical redox potentials of the components involved. DsbA contains the most oxidizing disulfide known, with a standard redox potential of –122 mV (13Zapun A. Bardwell J.C. Creighton T.E. Biochemistry. 1993; 32: 5083-5092Crossref PubMed Scopus (234) Google Scholar). DsbB contains two essential cysteine pairs, both of which have standard redox potentials that are substantially more reducing than that of DsbA according to most published reports. The Cys104– Cys130 pair of DsbB has been shown to react initially with DsbA (14Guilhot C. Jander G. Martin N.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 15Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar, 16Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the redox potential of this cysteine pair has been reported to be anywhere from –284 to –186 mV (17Grauschopf U. Fritz A. Glockshuber R. EMBO J. 2003; PubMed Scopus Google Scholar, K. Ito K. EMBO J. PubMed Scopus Google Scholar, K. Takahashi Y.H. Ito K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Bardwell J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (47) Google Scholar). for an of the flow of electrons from DsbA to redox potentials for the which is to the T. Yu M.W. Bardwell J.C. Yu C.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), are from mV (17Grauschopf U. Fritz A. Glockshuber R. EMBO J. 2003; PubMed Scopus Google to mV J. Bardwell J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (47) Google Scholar), with a intermediate and reported as well K. Ito K. EMBO J. PubMed Scopus Google Scholar, K. Takahashi Y.H. Ito K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in has a redox potential of mV Escherichia coli and and for Scholar). The potential for ubiquinone in is reported to be mV M. J. Biochem. PubMed Scopus Google Scholar). Although the redox potential of DsbB-bound ubiquinone has been it is to be substantially more oxidizing than DsbA, and is to the reaction the oxidation of DsbA the redox The by DsbB is reoxidized by the electron transport chain and the reaction. The of a complex has on the reaction mechanism of DsbB K. S. M. A. E. K. Ito K. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the bound ubiquinone The of the quinone is the membrane and is the in the is to the of the it was that which is in a of DsbB, from Cys130 upon DsbA binding. This to on the and the of the complex. Although this a in of the there kinetic the reactions proteins. We have rapid mixing kinetic measurements to the reaction DsbA and on results a kinetic for the DsbB reaction cycle. This study to the kinetic mechanism of an enzyme that the oxidizing of to disulfide it as the primary source of disulfide bonds in prokaryotes. and and DsbB proteins and according to published (5Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 7Bader M.W. Xie T. Yu C.A. Bardwell J.C. J. Biol. Chem. 2000; 275: 26082-26088Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), as and DsbB K. Takahashi Y.H. Ito K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y.H. K. Ito K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). DsbA was by with for at followed by DsbA was to be in a reduced as by with Biochem. 94: PubMed Scopus Google Scholar). Absorbance flow absorbance measurements on with a a (for a (for at in are in the shown are of a of to and the for The rates for at a concentration and are shown as one standard flow measurements on a with a An of nm nm spectral was in with a for experiments in at and absorbance measurements at for shown are of a of to and the for experiments using a according to the by J. Biol. Chem. Full Text PDF PubMed Google Scholar). DsbB was with DsbA and quinone ubiquinone ubiquinone in at and absorbance at 510 nm was The of the of the purple intermediate of DsbB is to the rate of enzyme and this was to a using A. was with using for and are reported as DsbA is for disulfide bond formation in proteins in the periplasm, which it by directly oxidizing substrate proteins exchange (3Frech C. Wunderlich M. Glockshuber R. Schmid F.X. EMBO J. 1996; 15: 392-398Crossref PubMed Scopus (50) Google Scholar, 4Zapun A. Creighton T.E. Biochemistry. 1994; 33: 5202-5211Crossref PubMed Scopus (88) Google Scholar). In to be in a catalytically reduced DsbA must be reoxidized by has been shown that reduced DsbA initially the disulfide of DsbB (14Guilhot C. Jander G. Martin N.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 15Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar, 16Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This binding is followed by a exchange reaction with a disulfide of The free thiolate of Cys44 is involved in a complex with the DsbB-bound quinone to a absorbance at ∼510 nm (11Inaba K. Takahashi Y.H. Fujieda N. Kano K. Miyoshi H. Ito K. J. Biol. Chem. 2004; 279: 6761-6768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We of this spectral intermediate to study the kinetic mechanism of by rapid mixing of of and are shown in upon of first is the rapid increase in absorbance at ∼510 which a at and with a slower rate to the initial is the absorbance at which substantially the first mixing but at a rate to that of the absorbance decay at 510 nm and for kinetic of absorbance at and 510 nm from the has a absorbance at nm ∼ m–1 that upon reduction. of DsbB-bound quinone be to a in absorbance at nm of and the absorbance was the of the in 2 is that the rise in absorbance at 510 nm reflects the of the DsbB and the subsequent decay both and 510 to quinone reduction. that this overall process is than reported in a published (11Inaba K. Takahashi Y.H. Fujieda N. Kano K. Miyoshi H. Ito K. J. Biol. Chem. 2004; 279: 6761-6768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This is most likely of in and likely that in at and the process be more rate the formation and decay of the purple intermediate of DsbB using measurements at 510 nm. upon mixing and is shown in The well to a for for yielded an suggesting that the purple intermediate in a The absorbance increase is of the formation of the intermediate at a rate of and the decay of this intermediate in two phases of and and The of the first and phases are and and with the phase only of the decay phase We a of experiments a of DsbA to which rates associated with the initial with DsbA, as to subsequent intramolecular reaction steps. The to and the rates DsbA shown in the rate of formation of the purple intermediate is highly dependent upon DsbA concentration (k1 × 105 m–1 This was the purple intermediate is to to an Cys44 of DsbB and the bound ubiquinone (11Inaba K. Takahashi Y.H. Fujieda N. Kano K. Miyoshi H. Ito K. J. Biol. Chem. 2004; 279: 6761-6768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Inaba K. Takahashi Y.H. Ito K. Hayashi S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 287-292Crossref PubMed Scopus (55) Google Scholar). DsbA, has been shown to initially the disulfide of DsbB (14Guilhot C. Jander G. Martin N.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 15Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar, 16Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (98) Google which in turn a exchange that results in the of the disulfide of DsbB a free thiolate at the purple intermediate only an intramolecular exchange reaction. Because it is an intramolecular reaction, this is be independent of DsbA concentration the rate of DsbA binding is slower than the intramolecular exchange reaction. The results in are suggestive of a exchange that is much than the rate of DsbA binding and formation of its disulfide This rapid disulfide exchange reaction of the process to the of The of DsbA at a much slower rate as by the of the absorbance increase at 510 the two subsequent decay phases show on DsbA concentration and that are intramolecular reaction steps. The decay phase with a rate of the of DsbA the decay at a rate of 0.3 The results in 2 suggest that the absorbance decay at nm the decay at 510 to the of quinone reduction. The observation that rates (k2 and on DsbA concentration is consistent with this utilized the absorbance of ubiquinone, which at 275 nm in the oxidized but is upon m–1 This a and by which to the rate of quinone reduction. We stopped flow absorbance measurements at 275 nm the same in the absorbance at 275 nm upon mixing DsbA and DsbB This is well by a and for for and with rates of and These rates are to the rates of decay of the purple that the rates of absorbance decay at 510 nm to the of quinone reduction. was for absorbance measurements at 510 the of the first phase is and followed by a decay phase on the known at 275 nm oxidized and reduced ubiquinone, absorbance suggest that of the bound quinone is reduced in this This may the of a of enzyme in the of DsbB-bound quinone is from that of free quinone in More kinetic in was be of the absorbance at 275 nm at protein experiments with DsbA suggested that the rates of absorbance decay at 275 nm and are independent of DsbA concentration as was for absorbance measurements at 510 nm. the at 275 which as a in absorbance of quinone reduction, reflects intramolecular reaction steps. This that quinone with DsbA, which is in with the model for DsbB The results suggest that the decay of absorbance at 510 and 275 nm is of of the DsbB-bound quinone. the that in protein absorbance to the at 275 experiments with a DsbA that one of the two essential it a exchange reaction with DsbB and support quinone reduction. a stable disulfide complex that is to a stable purple intermediate of DsbB (11Inaba K. Takahashi Y.H. Fujieda N. Kano K. Miyoshi H. Ito K. J. Biol. Chem. 2004; 279: 6761-6768Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), as in shown in rapid mixing of of DsbB with in a stable of the purple intermediate as by an absorbance increase at 510 nm. In to experiments with DsbA, subsequent absorbance decay was observed. In of absorbance at 275 nm showed that there was in upon mixing DsbB and This that in protein absorbance to the of a disulfide complex DsbB and are responsible for the absorbance at 275 nm in experiments with DsbA but that the in is of quinone reduction. Order of DsbB contains molecule of tightly bound ubiquinone In published it was at which the quinone to in the reaction. has been that DsbB the DsbA cysteine pair and that DsbB is oxidized by it is to the quinone an reaction in the overall this a of DsbB and of mixing In one that been with and with DsbA, and in the with a DsbA and and with DsbA, a rapid increase in absorbance at 510 nm followed by a slower decay phase was to results in previous The rate of absorbance increase was by of with that the rate of DsbA binding and the subsequent exchange reaction are dependent on the nature of the quinone The rate of decay of the absorbance at 510 nm the rate of quinone is slower than is with We have shown that the absorbance decay at 510 nm reflects the rate of quinone reduction. it that the of may to the quinone the of DsbB, with less than for We the formation of the purple intermediate is dependent on a quinone pre-bound to and DsbA, and with observe absorbance at 510 nm Absorbance at 275 nm the results at 510 of and DsbA followed by mixing with to results suggest that a pre-bound quinone on DsbB is for formation of the purple intermediate of DsbB absorbance at 510 as well as for efficient quinone at 275 nm). are why this be the the reaction may be a highly concerted process that requires quinone to be in disulfide exchange DsbA and DsbB and mixing DsbA and DsbB in the of quinone to an reaction. DsbA binding may the and quinone binding. This on the of the complex K. S. M. A. E. K. Ito K. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). binding to DsbB may be This and have and stopped flow experiments that to the that binding is are to initial that the reaction DsbA, DsbB, and ubiquinone is a highly concerted The highly concerted nature of the reaction why previous to dissect the reaction mechanism of DsbB by at individual cysteine pairs yielded seemingly paradoxical results. The results in that in for the reaction to DsbB must have a quinone bound to DsbA binding. The of the that this is in This DsbB and the of how reduced quinone is oxidized to a catalytically We see two the may with a and upon may from DsbB to be reoxidized by This be followed by a quinone binding the remain tightly bound to DsbB and be reoxidized by a quinone a complex. The that the quinone tightly bound in to the We are in studies at two DsbA is known to a upon which is of of the only by the disulfide bond J. A. M. Glockshuber R. Biochemistry. 1997; PubMed Scopus Google Scholar). DsbB a increase in upon reduction, and it is has been that this may be of of by quinone as it is reduced K. Takahashi Y.H. Ito K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the upon mixing reduced DsbA and oxidized DsbB are of from both proteins. We found that the increase of DsbB is than the upon DsbA the upon mixing and is a increase In an to dissect the of DsbB utilized a of DsbA in which the is with in a protein that and DsbB the increase was than the with DsbA We stopped flow measurements in an to the rate of DsbA oxidation the reaction cycle. This be by an initial in DsbB has reduced the quinone. We found that upon rapid mixing of and a in be The first phase is a but in followed by a and by a slower and increase in Because experiments than the absorbance measurements in and absorbance measurements at 510 nm to rates of absorbance reaction We found that rates in experiments to the phases in absorbance measurements the same suggesting that the for absorbance and are the stopped flow measurements a of DsbA that the first phase is highly dependent upon DsbA concentration the and and This that the initial in is of binding of DsbA the subsequent exchange which the free thiolate of in which DsbB with in a in suggesting that this is of binding of DsbA of the formation of the disulfide DsbA and DsbB, and of the oxidation of the disulfide of The observation that there are only two phases and phases with the rates of absorbance that DsbA oxidation and quinone at the same This is consistent with a highly concerted reaction, as has been suggested H. Beckwith J. EMBO J. PubMed Scopus Google Scholar). We kinetic measurements of upon mixing with DsbB Because is the kinetic in this is of in DsbB This showed the same of the with DsbA, a in followed by two phases in which the The overall of the is with the due to DsbA oxidation is with of that in the experiments with DsbA, the of DsbA oxidation are with the same rates as the of DsbB more likely of quinone This is suggestive of a highly concerted reaction in which DsbA oxidation and quinone are the of this study was to DsbB by the formation and decay of the to the rates for individual steps of the reaction with the overall rate of reaction We both a M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google and the by J. Biol. Chem. Full Text PDF PubMed Google to the kinetic and The the of the decay of an enzyme intermediate the purple species of upon reaction with an of substrate to a The of intermediate is of the of DsbB at shown in mixing DsbB with DsbA and to a of the purple intermediate for the first and this is followed by a decay in the as reduced DsbA is and the of This was to a which is shown in This yielded an of which is in with the published of M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The for is which is slower than and k2 but is than k3 The observation that is than k3 to that k3 a reaction is DsbA is in k3 it be to a reaction rate than 0.3 which reflects the rate of quinone reduction, is likely to be the overall rate-limiting is slower than k2 In are to of the nature of and as is a that is reduced more by DsbB with the rate of reduction. in experiments using the quinone by absorbance decay at 510 and 275 see is to the of Absorbance with DsbB a have that quinone is the rate-limiting in the DsbB reaction and that this is by a rapid intramolecular exchange reaction. quinone is the of DsbB with of redox potentials the rate of this on the other there is a rate-limiting that quinone as a exchange the rate of quinone on the redox potential of the quinone This has been in reaction G. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), which to the of the This that there is a other than charge transfer as a that the flow of for the of DsbB, and to that quinone is stopped flow absorbance measurements of the reaction of DsbB, with has a standard redox potential of mV Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), with mV for ubiquinone Escherichia coli and and for Scholar). Because is less oxidizing than ubiquinone, that DsbB more than ubiquinone there other rate-limiting quinone reduction. shown in this is is with mixing with DsbA, there is a rapid increase in the rate of which is to that mixing with DsbA and protein This the that the nature of the quinone DsbA binding the subsequent exchange event. The rate of absorbance decay reflects the rate of quinone on the other is from 2 for to 0.3 for The observation that the rate of quinone by decay at 510 is dependent on the redox potential of the quinone that quinone is rate-limiting in the overall reaction cycle. the rate of is to the rate of the decay phase in experiments with is to that the for the of this phase with and the of a and reaction in is of a of DsbB a quinone as other than ubiquinone have studied the of the DsbB reaction cycle in results support a kinetic model for DsbB in which DsbA binding is followed by a rapid exchange reaction. This results in the free thiolate of Cys44 and the of the This rapid exchange DsbA binding the reaction of reduced DsbA which at a much slower This DsbA binding to be tightly to quinone reduction. to be the rate-limiting in the overall reaction cycle. This is supported by the observation that of the quinone the rate of quinone reduction, the steps to quinone The mechanism of quinone was in the and this an and to to of this complex These studies the for the mechanism of quinone exchange to a catalytically We and for of the We for and for with