Title: Disulfide Bonds Are Generated by Quinone Reduction
Abstract: The chemistry of disulfide exchange in biological systems is well studied. However, very little information is available concerning the actual origin of disulfide bonds. Here we show that DsbB, a protein required for disulfide bond formation in vivo, uses the oxidizing power of quinones to generate disulfidesde novo. This is a novel catalytic activity, which to our knowledge has not yet been described. This catalytic activity is apparently the major source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that quinones get reduced during the course of the reaction. DsbB contains a single high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vivo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonuclease A is catalyzed by this system in a quinone-dependent manner. The disulfide isomerase DsbC is required to regain ribonuclease activity suggesting that the DsbA-DsbB system introduces at least some non-native disulfide bonds. We show that the oxidative and isomerase systems are kinetically isolated in vitro. This helps explain how the cell avoids oxidative inactivation of the disulfide isomerization pathway. The chemistry of disulfide exchange in biological systems is well studied. However, very little information is available concerning the actual origin of disulfide bonds. Here we show that DsbB, a protein required for disulfide bond formation in vivo, uses the oxidizing power of quinones to generate disulfidesde novo. This is a novel catalytic activity, which to our knowledge has not yet been described. This catalytic activity is apparently the major source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that quinones get reduced during the course of the reaction. DsbB contains a single high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vivo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonuclease A is catalyzed by this system in a quinone-dependent manner. The disulfide isomerase DsbC is required to regain ribonuclease activity suggesting that the DsbA-DsbB system introduces at least some non-native disulfide bonds. We show that the oxidative and isomerase systems are kinetically isolated in vitro. This helps explain how the cell avoids oxidative inactivation of the disulfide isomerization pathway. protein disulfide isomerase dithiothreitol denatured, reduced ribonuclease 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone 2,3-Dimethoxy-5-methyl-6-(10-bromo)-decyl-1,4-benzoquinone high performance liquid chromatography Much progress has been made in our understanding of how disulfide bonds are formed during protein folding in the cell. InEscherichia coli, a number of Dsb proteins catalyze the oxidation, reduction, and isomerization of disulfide bonds in newly exported proteins (for recent reviews see Refs. 1Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (238) Google Scholar, 2Debarbieux L. Beckwith J. Cell. 1999; 99: 117-119Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 3Glockshuber R. Nature. 1999; 401: 30-31Crossref PubMed Scopus (16) Google Scholar). Disulfide bond formation is crucial for the structure and stability of many of the proteins in which they are found. In prokaryotes, disulfides form in the rather oxidizing environment of the E. coli periplasm (1Rietsch A. Beckwith J. Annu. Rev. Genet. 1998; 32: 163-184Crossref PubMed Scopus (238) Google Scholar). The oxidizing power of the periplasm originates from the DsbA-DsbB system (4Bardwell J.C. McGovern K. Beckwith J. Cell. 1999; 67: 581-589Abstract Full Text PDF Scopus (846) Google Scholar, 5Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (364) Google Scholar, 6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). DsbA is a small soluble protein, which contains a thioredoxin-fold with a highly unstable disulfide bond. DsbA acts by transferring its active site disulfide bond very rapidly to folding proteins. This leads to the oxidation of the target protein and the reduction of the active site CXXC motif of DsbA (7Zapun A. Bardwell J.C. Creighton T.E. Biochemistry. 1993; 32: 5083-5092Crossref PubMed Scopus (235) Google Scholar, 8Grauschopf U. Winther J.R. Korber P. Zander T. Dallinger P. Bardwell J.C. Cell. 1995; 83: 947-955Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 9Martin J.L. Bardwell J.C. Kuriyan J. Nature. 1993; 365: 464-468Crossref PubMed Scopus (355) Google Scholar, 10Guddat L.W. Bardwell J.C. Martin J.L. Structure. 1998; 6: 757-767Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The inner membrane protein DsbB reoxidizes the active site CXXC motif of DsbA in vivo and in vitro (5Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (364) Google Scholar, 11Bader M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A similar system is responsible for forming disulfide bonds in the endoplasmic reticulum of eukaryotes (12Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Protein disulfide isomerase (PDI)1 is responsible for the net oxidation of protein thiols in vivo. A second protein termed Ero1p that reoxidizes PDI in vivo has been identified (13Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 14Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). The disruption of the ero1 gene leads to the accumulation of reduced PDI and a severe defect in the oxidation of the endoplasmic reticulum protein carboxypeptidase (12Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). PDI is also capable of isomerizing non-native disulfide bonds (15Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (663) Google Scholar). In E. coli the identification of an additional three Dsb proteins, DsbC, DsbD, and DsbG, led to the emergence of a second pathway that is responsible for the isomerization of incorrectly formed disulfide bonds in proteins with multiple disulfide bonds (16Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (197) Google Scholar, 17Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (171) Google Scholar, 18Andersen C.L. Matthey-Dupraz A. Missiakas D. Raina S. Mol. Microbiol. 1997; 26: 121-132Crossref PubMed Scopus (93) Google Scholar, 19Bessette P.H. Cotto J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). This system is particularly important for the heterologous expression of eukaryotic proteins containing multiple disulfide bonds such as urokinase (20Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar). In vivo, DsbA is mainly oxidized, explaining why it functions as an oxidant, whereas DsbC and DsbG are mostly found in their reduced states. This is a prerequisite for their function, since only a reduced isomerase can attack incorrectly formed disulfides (19Bessette P.H. Cotto J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar,20Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar). How are DsbG or DsbC kept reduced in the oxidizing environment of the bacterial periplasm and where does the oxidative power for the DsbA-DsbB system originate? The reduction of DsbC and DsbG depends on the presence of the inner membrane protein DsbD (19Bessette P.H. Cotto J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 22Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google Scholar). DsbD contains six essential cysteines and seems to connect the disulfide isomerization pathway with the reductive power of the cytosol of the cell (22Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google Scholar, 23Stewart E.J. Katzen F. Beckwith J. EMBO J. 1999; 18: 5963-5971Crossref PubMed Scopus (127) Google Scholar). It was found that the reduction of DsbC also depends on the presence of cytosolic thioredoxin or thioredoxin reductase (22Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google Scholar). It has been suggested that thioredoxin keeps the two cytosolic cysteines reduced which in a series of intramolecular disulfide exchange reactions reduce the CXXC motif of DsbD which then reduces DsbG or DsbC (22Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google Scholar). The existence of DsbD, however, does not explain why the oxidative DsbA-DsbB system is apparently incapable of oxidizing and thus inactivating the isomerization pathway. Regarding the oxidative pathway, it could be shown that components of the electron transport chain serve as immediate electron acceptors of DsbB (6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 24Kobayashi T. Ito K. EMBO J. 1999; 18: 1192-1198Crossref PubMed Scopus (92) Google Scholar). By reconstituting the DsbA-DsbB system with purified components in vitro, we were able to show that ubiquinone serves as immediate electron acceptor of DsbB (6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Ubiquinones are then reoxidized by terminal oxidases such as cytochrome bd andbo oxidase which finally transfer electrons onto oxygen. The observation that quinones are electron acceptors of DsbB demonstrates why disulfide bonds still form under anaerobic conditions (6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Menaquinones whose synthesis is up-regulated upon oxygen depletion (25Wallace B.J. Young I.G. Biochim. Biophys. Acta. 1977; 461: 84-100Crossref PubMed Scopus (209) Google Scholar) are able to reoxidize DsbB in vitro. Menaquinones are then reoxidized by anaerobic reductases such as fumarate reductase. Here, we characterize the reoxidation of DsbB by quinones. We report evidence that ubiquinone gets reduced upon DsbB-mediated oxidation of DsbA, and we propose a specific binding site for ubiquinones that we titrated with externally added ubiquinone. We further show that the DsbA-DsbB-quinone system efficiently reoxidizes reduced RNase A, but that RNase activity is only regained when the disulfide isomerase DsbC is present. DsbA and DsbC were purified essentially as described before (11Bader M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 30Darby N.J. Raina S. Creighton T.E. Biochemistry. 1998; 37: 783-791Crossref PubMed Scopus (41) Google Scholar). DsbB was purified from membranes prepared from the DsbB overexpression strain WM76. The membranes were solubilized in 1% n-dodecyl maltoside. The His-tagged DsbB was then bound to a nickel-nitrilotriacetic acid column that had been equilibrated with 50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 0.02% n-dodecyl maltoside by passing the solubilized membranes over the column at a flow rate of 0.2 ml/min. The column was washed with the same buffer containing 50 mm imidazole. DsbB was eluted with a linear imidazole gradient ranging from 50 to 300 mm. Fractions containing DsbB were pooled and loaded directly onto a hydroxyapatite column equilibrated with 50 mm sodium phosphate, pH 6.2, 100 mm NaCl, and 0.1% n-dodecyl maltoside. DsbB was eluted from the column with a linear gradient in a buffer that contained 300 mm NaCl, 0.1% n-dodecyl maltoside, and sodium phosphate ranging in concentration from 50 to 500 mm. Fractions containing purified DsbB were concentrated to 5 mg/ml and dialyzed versus 10 mm Hepes, pH 7.5, 50 mm NaCl. The protein was stored at −70 °C without loss of activity for >6 months. The DsbB concentration was determined after reduction of protein-bound quinone with NaBH4 using the extinction coefficient of ε276 = 46.5 mm−1. DsbC and DsbA were reduced by incubation in 10 mmDTT for 20 min on ice. Proteins were purified from DTT by PD-10 (Amersham Pharmacia biotech) gel filtration in 20 mm Hepes, pH 7.5, 0.5 mm EDTA. The thiol content was measured with DTNB as described (26Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Crossref PubMed Scopus (1090) Google Scholar). DsbA and DsbC were >95% reduced and stored at −70 °C until use. DsbB activity was followed in 50 mm sodium phosphate, pH 6.0, 300 mm NaCl, 0.1%n-dodecyl maltoside at 25 °C. Reoxidation of DsbA was measured with a Hitachi fluorescence spectrophotometer as described before (11Bader 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 reduction of ubiquinone analogues was measured photometrically (Beckman). Q0C10 reduction was followed at 275 nm with an extinction coefficient of 12.25 mm−1. The reaction was started by the addition of a small volume of DsbB. The concentration of DsbB was between 0.5 and 5 nm and was linear to the initial velocity over this range of enzyme concentration. For steady state kinetics, initial rates were derived from the linear decrease of either fluorescence of DsbA or ubiquinone. The rates were transformed into nm DsbA/s or nm quinone/s and plotted against the concentration of substrate. The data were fitted to a hyperbola, and V max and K m values were obtained from the fit. Absorbance of DsbB was recorded from 240 to 390 nm in 50 mm sodium phosphate, pH 6.0, 300 mm NaCl, 0.1% n-dodecyl maltoside. Ubiquinones were reduced by adding few grains of solid sodium borohydride to the cuvette and mixing thoroughly. After 5 min incubation at room temperature, the reduced spectrum was recorded from 240 to 390 nm. Employing an absorption coefficient of Δε275 = 12.25 mm−1, we calculated the amount of bound ubiquinone. The quinone species bound to DsbB was identified and quantified by high performance liquid chromatography (HPLC). Methanol (−20 °C) was added to 1.4 ml of DsbB (4.4 mg/ml) to yield a final volume of 10 ml and vortexed immediately. Ubiquinone was extracted with 4 × 15 ml of hexane. The sample was dried by evaporation of the solvent and dissolved in 5 ml of diethyl ether. The sample was dried again, dissolved in 0.5 ml of 95% ethanol, filtered through a 0.2-μm membrane, and loaded onto a Microsorb-MV® reverse phase column (C8, 5 μm). Coenzymes Q1, Q2, Q8, and Q10 were used as standard quinone compounds for HPLC. Coenzyme Q8 was extracted from E. colimembranes by following a published procedure (27Redfearn E.R. Methods Enzymol. 1967; 10: 381-384Crossref Scopus (78) Google Scholar). Ubiquinones were eluted from the column with a linear gradient ranging from 90 to 100% methanol (v/v). The flow rate was 0.8 ml/min. The ubiquinone species bound to DsbB was identified and quantified based on the retention time and peak area of known standard coenzyme Qs. Purified DsbB was washed with 10 volumes of titration buffer containing 50 mm K+/Na+phosphate, pH 7.4, 1.0% sodium cholate prior to quinone titration. This was done because sodium cholate generally improves the signal obtained during quinone titrations over that obtained in the presence of dodecyl maltoside. Indeed no signal could be detected for DsbB in the presence of 0.1% dodecyl maltoside, but a good signal was detected in the sodium cholate detergent. Titration experiments were performed in a total volume of 1 ml at a DsbB concentration of 0.44 mg/ml (22 μm). 2,3-Dimethoxy-5-methyl-6-(10-bromo)-decyl-1,4-benzoquinone (Q0C10Br) was added stepwise in 1-μl volumes from a 5 mm stock solution. After the addition of 1 μl of quinone solution, the sample was incubated for 15 min at room temperature, and the spectra were recorded from 240 to 340 nm. The absorbance change at 280 nm upon addition of Q0C10Br was plotted against the concentration of quinone added to the cuvette. A titration experiment in the absence of DsbB was performed as a control and showed basically the same change in absorbance for each μl of added quinone. Ribonuclease A was incubated in 100 mm Tris-HCl, pH 8.0, 6 m guanidine hydrochloride, 120 mm DTT, 0.2 mm EDTA for 1.5 h at 37 °C. The buffer was exchanged to 0.1% acetic acid by PD-10 gel filtration. The column was equilibrated in 0.1% acetic acid. Reduced denatured ribonuclease was quantified by using ε275.5 = 9.3 mm−1, whereas an absorbance coefficient of ε275.5 = 9.8 mm−1 was used for the native protein. Oxidative refolding was initiated by dilution of drRNase into DsbB assay buffer (50 mm sodium phosphate, pH 6.0, 300 mm NaCl, 0.1% n-dodecyl maltoside). The buffer also contains 0.1 μm DsbB, 50 μm Q-1, and 0.1–1.0 μm oxidized DsbA. The final concentration of drRNase was 10 μm. Oxidative refolding was monitored by following the reduction of Q0C10 at 275 nm. Q0C10 reduction was dependent on the presence of catalytic quantities of oxidized DsbA. To test whether ribonuclease gained catalytic activity after exposure to the DsbA-DsbB system, aliquots were taken and diluted 1:10 into the same buffer containing 5 mm cCMP. Native RNase catalyzes the hydrolysis of cCMP that can be monitored at 296 nm (Δε296 = 0.19 mm−1). Activity was monitored in the presence or absence of the reduced disulfide isomerase DsbC (10 μm final concentration). Native RNase served as a positive control under the same assay conditions. The ultimate source of oxidizing equivalents for the formation of disulfide bonds in the prokaryote E. coli originates in the electron transport system (6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 24Kobayashi T. Ito K. EMBO J. 1999; 18: 1192-1198Crossref PubMed Scopus (92) Google Scholar). By reconstituting the DsbA-DsbB systemin vitro, we demonstrated that DsbB is reoxidized by quinones (6Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Our data suggest that ubiquinones function as the electron acceptors of DsbB under aerobic growth while under anaerobic conditions menaquinones reoxidize DsbB. Enzymatic activity of DsbB can be measured by following the reoxidation of DsbA which results in a decreased relative fluorescence of DsbA upon oxidation (11Bader M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Apparently, quinones serve as the second substrate of DsbB, and it can be predicted that quinones should get reduced during the course of the reaction. To demonstrate this, we decided to monitor the reaction by simply following the absorption change between ubiquinone and its corresponding ubiquinol (Δε275 = 12.25 mm−1). Fig.1 shows that DsbB efficiently reduces Q0C10 as measured by the absorbance change at 275 nm. No reduction of ubiquinone is observed in the absence of DsbB. Only when DsbB is added to the reaction mixture in catalytic amounts did we observed reduction of ubiquinone. Enzymatic activities were derived from the initial slopes of absorbance decrease. For ubiquinone reduction we observed an activity of 278 nmol of ubiquinone per nmol of DsbB per min. This activity agrees well with the activity derived from the fluorescence assay measured under the same conditions (data not shown). Thus, this new assay can be employed to determine the activity of DsbB. In order to characterize the ubiquinone reductase activity of DsbB, we measured initial velocities of ubiquinone reduction at various concentrations of Q0C10. The concentration of DsbA was held constant at 20 μm, whereas the concentration of the quinone was varied (Fig.2 A). To obtain kinetic constants the curves were fit to the Michaelis-Menten equation. When Q0C10 was used as a substrate the apparentK m was 2.0 μm, whereasV max was 5.0 nmol of DsbA per nmol of DsbB/s. The concentration of DsbA was at 20 μm (0.42 mg/ml), and a less than 15% increase in velocity was observed at 40 μm reduced DsbA (data not shown). This suggests that at a concentration of 20 μm the reduced DsbA is close to saturating conditions. To verify this further, we measured theK m for DsbA under saturating concentrations of Q0C10. The K m value for DsbA was previously reported to be 10–13 μm (11Bader M. Muse W. Zander T. Bardwell J. J. Biol. Chem. 1998; 273: 10302-10307Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 28Jonda S. Huber-Wunderlich M. Glockshuber R. Mossner E. EMBO J. 1999; 18: 3271-3281Crossref PubMed Scopus (68) Google Scholar). This value was derived from measurements of DsbB activity in membranes. An apparent K m value for the highly purified system was determined to be 3.1 μm (Fig. 2 B). This value was measured under saturating concentrations of quinone (25 μm) and in the presence of 0.1% dodecyl maltoside. The apparent k cat/K m values for ubiquinone is 3·106m−1 s−1, making quinones very specific substrates for DsbB. To our knowledge, DsbB has an undescribed enzymatic activity; it catalyzes the formation of a disulfide bond by the reduction of ubiquinone. The observation that DsbB catalyzes the reduction of quinones led us to analyze the quinone-binding properties of DsbB. We first decided to test if DsbB contains a quinone bound after purification. Oxidized quinones can be detected by a change in absorption at 275 nm upon reduction by addition of sodium borohydride. Ubiquinones show an absorbance peak at 275 nm, which decreases upon reduction of the quinone to the quinol. Fig.3 shows the UV spectra of purified DsbB before and after the addition of sodium borohydride (NaBH4). We observed an 11% decrease in the absorption at 275 nm after NaBH4 was added to the cuvette. We attributed this change in absorbance to the reduction of a ubiquinone bound to DsbB. The addition of the NaBH4 did not change the absorbance of the buffer blanked against water or DsbA, which served as a control (data not shown). From this absorbance change and given that the absorbance coefficient of coenzyme Q-8 is 12.25 mm−1 we calculated that the purified DsbB contains 0.5 molar eq of bound quinone. In order to obtain additional proof for binding of ubiquinone to DsbB, we extracted DsbB with hexane, which should denature the protein and extract any bound quinones. The extract was analyzed by HPLC. The comparison of the elution profile of the DsbB extract (Fig.4 a) with the elution profiles from known quinone standards (c and d) indicates that the sample extracted from DsbB contains coenzyme Q-8. Mixtures of the DsbB extract and purified coenzyme Q-8 migrate as a single peak (Fig. 4 b). The identification of coenzyme Q-8 as the DsbB-bound quinone agrees very well with the fact that this quinone is the predominant quinone species present in E. coli membranes under aerobic growth conditions (25Wallace B.J. Young I.G. Biochim. Biophys. Acta. 1977; 461: 84-100Crossref PubMed Scopus (209) Google Scholar). From the peak area we calculated a molar ratio of 0.6 mol of coenzyme Q-8 (ubiquinone Q0C40) bound per mol of DsbB, consistent with the value derived from the difference spectra shown in Fig. 1. We wanted to test to see how many high affinity quinone-binding sites were present in DsbB. Quinone-binding sites in proteins can be titrated by addition of external quinone, with the binding being followed by an absorbance change of the quinone upon binding to the protein (35Shenoy S.K., Yu, L. Yu C. J. Biol. Chem. 1999; 274: 8717-8722Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Fig.5 demonstrates that the presence of DsbB strongly affects the spectral properties of externally added Q0C10Br. Q0C10Br has been shown to be a better ubiquinone analogue for the study of protein-quinone-interaction (36Yu C.A. Yu L. J. Biol. Chem. 1982; 257: 6127-6131Abstract Full Text PDF PubMed Google Scholar). The different spectral properties of Q0C10Br in the presence of DsbB indicate the transfer of Q0C10Br from an aqueous to a more hydrophobic environment. We attribute this absorbance change to the binding of ubiquinone to DsbB. The absorbance change of Q0C10Br upon interaction with DsbB reaches a saturation level (closed circles). After addition of 20 μm, Q0C10Br showed the spectral properties that are typically observed for ubiquinone in the aqueous buffer control (open circles). The DsbB concentration was 22 μm. By taking into account that the protein contained 0.3 mol of bound ubiquinone after the detergent exchange, the titration with external quinone provides evidence that the quinone site of DsbB can be titrated to a 1:1 molar ratio. This demonstrates that DsbB possesses a single, highly specific quinone-binding site. DsbA is a rather nonspecific, but powerful, oxidant capable of acting on many folding proteins, including eukaryotic proteins expressed in E. coli. DsbB presumably has a much more limited substrate specificity. It functions to reoxidize DsbA in vitro and in vivo. The severe disulfide defect present in strains that lack DsbA is good evidence that DsbB is incapable of directly oxidizing folding proteins in vivo (5Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (364) Google Scholar). Our ability to measure directly DsbB activity by its ubiquinone reductase activity now allows us to test directly the rate at which DsbB can oxidize unfolded proteins and small molecule thiol-containing compounds. As a potential substrate for DsbB we chose ribonuclease A, which contains four disulfide bonds in its native state. The oxidative refolding of RNase has been well studied making this protein an excellent model substrate for our in vitro system (29Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 613-619Crossref PubMed Scopus (359) Google Scholar). We tested if catalytic quantities of DsbB (0.1 μm) were capable of quinone reduction after the addition of 10 μm denatured, reduced RNase A. We were, however, not able to detect any DsbB-catalyzed quinone reduction in the absence of DsbA (TableI). Only after addition of a catalytic amount of oxidized DsbA (0.1 μm), a decrease in absorbance at 275 nm was observed (Fig.6). This provides good biochemical evidence that DsbB is not capable of directly oxidizing RNase A under conditions where DsbA can function. This is further evidence that DsbA acts as the direct donor of disulfides during protein refolding. Reduced DsbA generated from the oxidation of RNase serves as the substrate of DsbB, which in turn reduces quinone. The reduction of quinones was measured as a decrease in absorbance at 275 nm. We followed this absorbance change until the reaction was complete in order to determine the stoichiometry of the reaction (Fig. 6). The total amount of reduced ubiquinone was determined and plotted against the initial concentration of ubiquinone. The titration curve shows that the reduction of 40 μm quinones is necessary to oxidize completely 10 μm RNase A. Since RNase A contains 4 disulfides, it appears that one quinone gets reduced for every disulfide bond formed. That ribonuclease was completely oxidized was verified by 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate trapping and non-reducing SDS-polyacrylamide gel electrophoresis (data not shown).Table IDsbB specifically reoxidizes DsbAPotential DsbB substrateConcentrationUbiquinoneActivityμmdrRNase1020<0.5GSH10020<0.5DsbC, reduced1020<0.5DsbA, reduced1020243No DsbB activity was detected with drRNase, GSH, and reduced DsbC as substrates at DsbB concentrations up to 250 nm. We determined the lower limit of detection of DsbB activity as 0.5 nm DsbB under standard assay conditions. Thus, reduced DsbA is oxidized at a rate at least 500-fold faster than the other three substrates tested. Open table in a new tab No DsbB activity was detected with drRNase, GSH, and reduced DsbC as substrates at DsbB concentrations up to 250 nm. We determined the lower limit of detection of DsbB activity as 0.5 nm DsbB under standard assay conditions. Thus, reduced DsbA is oxidized at a rate at least 500-fold faster than the other three substrates tested. DsbA is regarded by some workers (20Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar, 30Darby N.J. Raina S. Creighton T.E. Biochemistry. 1998; 37: 783-791Crossref PubMed Scopus (41) Google Scholar), including the authors of th