Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

Abstract

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Many extracytosolic proteins contain disulfide bonds. Recent studies established that multiple cellular factors participate in the pathway of disulfide bond formation in Escherichia coli (1, 2). DsbA, a periplasmic protein, is the primary catalyst of protein disulfide bond formation (3–6). While DsbA’s role is to introduce disulfide bonds to a newly synthesized and exported protein, DsbC, another periplasmic factor (7), acts to isomerize the disulfide bonds (8–10).

DsbA has a redox active site, Cys-30-Pro-His-Cys-33, and the Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. The Cys-30 residue of DsbA has unusually low pKa value and the active site of this protein has very high redox potential (11, 12). The strong oxidizing power of DsbA means that it is prone to reduction. Indeed, the reduced state of DsbA was shown to be thermodynamically more stable than the oxidized state (13–15). We showed previously that, in spite of these properties, DsbA is kept essentially all oxidized in vivo (16). Thus, whenever DsbA is reduced, as the obligatory consequence of its catalytic function, it is immediately reoxidized. It should be noted that for assessing the in vivo redox states of this protein, it was essential to “freeze” the in vivo state by a direct treatment of the culture with trichloroacetic acid, thereby denaturing all cellular proteins; otherwise, some artificial reduction can occur for native DsbA after disruption of the cell (16).

What is then the mechanism that assures rapid oxidation of DsbA in the cell? DsbB, an integral membrane protein, was suggested to reoxidize DsbA (17). In support of this notion, DsbA remains reduced in the dsbB-disrupted mutant (16, 17), and a DsbA variant lacking Cys-33 forms a DsbA–DsbB intermediate complex, in which Cys-30 of DsbA and Cys-104 of DsbB are disulfide-bonded (18–20). We proposed that the active site cysteines of DsbA are oxidized by Cys-104–Cys-130 of DsbB, followed by an intramolecular transfer of disulfide from Cys-41–Cys-44 (20). However, it is totally unknown how the oxidative state of the DsbA/DsbB system is maintained. In other words, what might accept electrons that flow from newly synthesized secretory proteins via DsbA and DsbB.

For aerobically growing cells, the air oxygen might be the ultimate source of the oxidizing power, and there is a possibility that the respiratory electron transfer chain is involved in the pathway of oxidation of DsbA. We now addressed this question by examining redox states of DsbA in mutants defective in the components of the respiratory chain. Experiments using hemA mutant cells as well as ubiA-menA mutant cells showed that DsbA remains reduced during growth under the conditions of protoheme deprivation as well as under the conditions of deprivation of ubiquinone and menaquinone. Under these conditions, disulfide bond formation of β-lactamase did not occur with normal efficiency. Intracellular state of DsbB was also affected under these conditions; it was converted into a form of DsbA–DsbB disulfide-linked complex. These results suggest that the functional electron transfer chain is involved in the disulfide bond formation pathway.

MATERIALS AND METHODS

E. coli Strains and Plasmids.

Strains used were CU141 (MC4100/F′lacI^Q lacPL8 lacZ⁺ Y⁺ A⁺ pro⁺; ref. 21), SS141 (CU141, dsbB::kan5; refs. 17 and 18), LE392 (22), H500 (LE392, ΔhemA::kan; ref. 23), AN387 (ubi⁺ men⁺; ref. 24), and AN384 (ubiA420 menA401; ref. 24). TA58 was a dsbB::cat derivative of AN387, and constructed by first transforming a recD strain with a DNA fragment carrying dsbB that had an insertion of a cat determinant (a HaeII fragment from pACYC184) in the HpaI site and then transducing the insertion by P1 transduction. Strain H500 hemP was one of the hemin-permeable derivatives of H500 described by Nakayashiki and Inokuchi (25). pSS39 was a plasmid in which dsbB was cloned under the lac promoter (18). pSS1 had a bla-phoA fusion gene from pKY192 (4) cloned into a lac promoter vector, pNO1575 (26). This plasmid encodes normal β-lactamase as well as a β-lactamase (amino-terminal 5 residues of the mature part)-PhoA fusion protein.

Media and Growth Conditions.

L-medium contained 10 g tryptone, 5 g yeast extract, 5 g NaCl, and 1.7 mmol NaOH per liter. In most experiments it was further supplemented with 0.4% glucose. Potassium phosphate buffer (pH 7.5) was also included at a final concentration of 0.09 M when indicated. 5-Aminolevulinic acid (ALA; 50 μg/ml), hemin chloride (10 μg/ml), p-hydroxybenzoate (PHB; 1 mM), and chloramphenicol (100 μg/ml) were added, as indicated. Cells were grown aerobically with shaking at 37°C.

Determination of Redox States of DsbA.

Redox states of DsbA in vivo were assessed as described (16, 20), except that free SH groups were modified by 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (AMS; ref. 27) instead of iodoacetamide. Briefly, whole cell proteins were precipitated by direct treatment of a culture with final 5% of trichloroacetic acid to avoid any subsequent reduction of DsbA after cell disruption. Protein precipitates were collected by centrifugation, washed with acetone, and dissolved in freshly prepared solution containing 1% SDS, 50 mM Tris⋅HCl (pH 7.5), and 15 mM AMS. Modification with iodoacetamide was done as described by Pollitt and Zalkin (28). Proteins were then separated by 12.5% SDS/PAGE (29) without using any reducing agent. DsbA was visualized by Western blot analysis (5, 21).

Preparation of Anti-DsbB Antibodies.

A synthetic peptide with amino acid sequence corresponding to the carboxyl-terminal 14 residues of DsbB (17) with an additional cysteine at the amino terminus was coupled with keyhole limpet hemocyanin and injected into a rabbit. Serum was purified by affinity binding to thiopropyl Sepharose-coupled antigen.

RESULTS

An Improved Method for Examination of the In Vivo Redox States of DsbA.

The redox states of DsbA in vivo can only be assessed accurately after acid denaturation of cellular proteins (16). Thus, trichloroacetic acid is added directly to the cultural medium to denature and precipitate whole cell proteins, which are then dissolved in high-pH SDS solution containing a SH-blocking reagent (28). Reduced and oxidized forms of DsbA are then separated by SDS/PAGE. This precaution is needed because the native DsbA protein is subject to artificial reduction after cell disruption (16). We improved the electrophoretic separation of the reduced and the oxidized DsbA molecules by modifying free SH groups with AMS, instead of iodoacetamide used conventionally (see Fig. 1, lane 3). Previous reports showed that the AMS-modification markedly retards electrophoretic migrations of proteins in SDS/PAGE (27, 30). We were thus able to achieve unequivocal separation of oxidized and reduced (and AMS-modified) forms of DsbA molecules (Fig. 1, lanes 1 and 2). Immunoblotting of the AMS-treated cellular proteins confirmed that DsbA in wild-type cells is totally oxidized (Fig. 1, lane 4) whereas that in the dsbB-disrupted cells is totally reduced (Fig. 1, lane 5).

Figure 1.

Separation of reduced and oxidized DsbA. Lanes 1–3: Purified DsbA (5) was diluted into SDS-sample buffer (29) without reducing agent (lane 1), or first reduced with 17 mM DTT at 37°C for 10 min, precipitated with trichloroacetic acid, washed with acetone, and dissolved in either 1% SDS, 50 mM Tris⋅HCl (pH 7.5) containing 15 mM AMS (lane 2) or SDS, Tris⋅HCl, EDTA, iodoacetamide (ref. 28; lane 3). About 500 ng of DsbA was electrophoresed. Lanes 4 and 5: Strains CU141 (wild-type; lane 4) and SS141 (dsbB::kan5; lane 5) were grown in L medium, and whole cell proteins were precipitated with trichloroacetic acid, washed with acetone, and dissolved in 1% SDS, 50 mM Tris⋅HCl (pH 7.5), 15 mM AMS. Samples corresponding to about 1.5 × 10⁷ cells were electrophoresed. DsbA was visualized by Western blot analysis.

The hemA Mutant Accumulates the Reduced Form of DsbA Under Growing Conditions.

The hemA gene of E. coli encodes glutamyl-tRNA reductase for the commitment reaction of protoheme and siroheme biosynthesis (31, 32). Its disruption causes apparent ALA auxotrophy. The hemA::kan cells (23) grew in L-broth-glucose without supplement of ALA up to about 3 × 10⁸/ml and then stopped growing. Curiously, cells grew again to a similar density when the stationary cells prepared as above were reinoculated into a fresh medium of the same composition. Although DsbA remained oxidized during the first cycle of cell growth in the absence of ALA, it became reduced during the second cycle of growth in the same medium. The reduced DsbA was detected as the AMS-modified (Fig. 2A, lane 1) or iodoacetamide-modified (Fig. 2A, lane 3) forms. It should be noted that whenever samples were withdrawn after cessation of cell growth, DsbA was observed as the oxidized form (Fig. 2A, lane 2). DsbA was always oxidized in the isogenic hemA⁺ strain (Fig. 2A, lane 4).

Figure 2.

DsbA is reduced during growth under the protoheme-deprived conditions. (A) Accumulation of reduced DsbA. For lanes 1–4, Strains H500 (ΔhemA::kan; lanes 1–3) and LE392 (isogenic hemA⁺ strain; lane 4), pregrown in L-glucose medium supplemented with ALA, were inoculated into the same medium without ALA. After overnight shaking, cells were diluted into the same medium, and samples for lanes 1, 3, and 4 were withdrawn at turbidity of 45 (Klett colorimeter with no. 54 filter), while sample for lane 2 was withdrawn after growth cessation (at about 77 Klett reading). For lanes 5–7, H500 was grown in L-glucose-ALA medium that was further supplemented with potassium phosphate (pH 7.5), washed with the same medium but without ALA and grown in the ALA-free medium for 370 min (lane 5), 420 min (lane 6), and 490 min (lane 7), with a dilution at 280 min. The sample for lane 3 was treated with iodoacetamide, whereas all the other samples were treated with AMS. (B) Reoxidation of DsbA after addition of a down-stream intermediate (ALA) or the final product (hemin) of the heme biosynthetic pathway. Strains H500 (ΔhemA; Upper) and its hemin-permeable derivative, H500 hemP (Lower), were grown for two cycles in the absence of ALA as described in A (lane 1), and ALA or hemin chloride, respectively, was added, followed by sampling at 0 (lane 1), 1 (lane 2), 3 (lane 3), 10 (lane 4), and 20 (lane 5) min. Samples for lane 6 had been mock treated with water for 20 min.

In the absence of heme biosynthesis, respiratory cytochromes will remain in their apo-forms without activity (33). Since cells should be able to utilize glucose as an energy source even in the absence of respiration, the early cessation of cell growth after the removal of ALA should have been a secondary consequence of the lack of respiration. Alternatively, a defect in the formation of catalase or some other enzymes might have been responsible. We found that a lowering of the medium pH, to below 5, was responsible for the early growth cessation, consistent with the growth resumption upon reinoculation into a fresh medium. When phosphate buffer (pH 7.5) or Hepes buffer (pH 7.5) was supplemented to the broth medium, the cells continued to grow albeit at a reduced rate to an almost normal stationary cell density. The buffered broth enabled a simple ALA removal experiment; as the cell growth continued in the absence of ALA, DsbA became progressively more reduced (Fig. 2A, lanes 5–7). At stationary phase, DsbA was oxidized again (data not shown).

When ALA was added back to the heme-deprived culture, DsbA was converted to the oxidized form again (Fig. 2B, Upper). This conversion took 10–20 min. ALA, which accelerated the cell growth, should have exerted its effect via the recovery of heme biosynthesis, rather than the mechanism considered below for general cessation of protein synthesis. Recovery of the DsbA oxidation was also observed when hemin chloride was added to the culture of a hemin-permeable derivative of the hemA mutant (Fig. 2B, Lower). This result indicates that it was the lack of protoheme biosynthesis, rather than that of siroheme biosynthesis, that was responsible for the accumulation of the reduced form of DsbA.

The hemA Mutant Accumulates the Reduced Form of β-Lactamase.

The accumulation of the reduced form of DsbA is expected to result in an impairment of disulfide bond formation of secretory proteins. The hemA mutant, when grown in the ALA-free medium, indeed accumulated reduced form of β-lactamase synthesized from a plasmid (Fig. 3). Thus, normal synthesis of heme molecules is required for cells to support efficient disulfide bond formation.

Figure 3.

Effects of heme deprivation on disulfide bond formation of β-lactamase. Strains LE392 (wild-type; lane 1) and H500 (ΔhemA; lanes 2–5) were transformed with pSS1 carrying bla. Cells were grown on L-glucose-ALA medium supplemented with potassium phosphate (pH 7.5), washed, and grown in the ALA-free medium for 8 (lanes 1), 0 (lane 2), 6 (lane 3), 12 (lane 4), and 24 (lane 5) hr with appropriate dilutions. Samples were treated with iodoacetamide, electrophoresed, and visualized with anti-β-lactamase serum (obtained from 5′ → 3′).

DsbA Is Also Reduced During Growth Under Deprivation of Ubiquinone and Menaquinone.

The results presented above suggest strongly that some heme-containing protein(s), such as (a) cytochrome(s), is required for the efficient oxidation of DsbA. To address whether the functional electron transfer system is required, we used the ubiA-menA double mutant strain (24). These quinones are of vital importance for respiration of the E. coli cell (24). The ubiA gene encodes PHB octaprenyltransferase for ubiquinone biosynthesis (34) whereas the menA gene encodes 1,4-dihydroxy-2-naphthoate octaprenyltransferase for menaquinone biosynthesis (35). The enzymatic defect of the ubiA mutant used can be alleviated by an inclusion of PHB in the medium, because the mutant enzyme has a lowered affinity for this substrate (34). We found again that growth of this mutant can be improved by an inclusion of a buffer in the broth. Thus, cells were first grown in the buffered broth in the presence of glucose and PHB, and then washed to remove PHB. The mutant cells continued to grow at a nearly normal rate for about 2 hr and then at a slower rate (Fig. 4A, •). DsbA was found to become reduced after the cells entered the slower growth phase (Fig. 4B, lanes 4–6). When PHB was added back to restore the enzyme activity, DsbA was oxidized again (Fig. 4C). The redox state of DsbA was affected much less pronouncedly in the ubiA single mutant strain (data not shown).

Figure 4.

DsbA is reduced under the ubiquinone and menaquinone-deprived conditions. (A) Growth curves. Strains AN387 (wild-type; ○) and AN384 (ubiA420 menA401; •) were grown first in L-glucose medium supplemented with potassium phosphate buffer and PHB, washed with the same medium without PHB, and grown in the PHB-free medium with a dilution as indicated by the gap. Turbidity of the culture was measured by a Teitech (Tokyo) Photometer 518, whose reading of 0.1 corresponded to OD₆₀₀ of about 0.3. (B) Accumulation of reduced DsbA. Samples of the cultures shown in A were withdrawn at the points indicated by arrows (a and b for AN387; 1–6 for AN384), and examined for redox states of DsbA after AMS modification (the lane numbers correspond to the sampling numbers). (C) Reoxidation of DsbA after recovery of the ubiA function. PHB was added to the AN384 culture at the point indicated by arrow 4 in A and samples were taken at 0 (lane 1), 1 (lane 2), 3 (lane 3), 10 (lane 4), or 20 (lane 5) min. The sample for lane 6 had been mock treated with the solvent (ethanol) for 20 min.

A Respiratory Chain-Independent Pathway May Suffice to Keep DsbA Oxidized in the Absence of Active Protein Synthesis.

Like the hemA mutant, the ubiA-menA mutant failed to accumulate reduced DsbA at a stationary growth phase. Thus, accumulation of reduced DsbA only occurs when cells are growing under the conditions of heme or quinone deprivation. The general conformational stability of disulfide-bonded proteins suggests that only newly synthesized proteins before their final folding can be a substrate of DsbA (10, 36). In the absence of protein synthesis, DsbA finds few newly synthesized secreted proteins, and, hence, few chances to be reduced. This seems to explain the disappearance of the reduced form upon growth cessation.

To examine whether inhibition of protein synthesis causes oxidation of DsbA, we added chloramphenicol to the culture in which the hemA mutant had undergone sufficient growth in the absence of ALA to accumulate reduced DsbA. It was found that the addition of chloramphenicol caused gradual oxidation of DsbA. After 40 min, DsbA became largely oxidized (Fig. 5B). The control culture that received H₂O did not show any conversion of the reduced DsbA (Fig. 5A). Chloramphenicol caused oxidation of DsbA in the dsbB-disrupted strain as well (Fig. 5C, lane 4), although the response was much delayed as compared with the effect on the hemA mutant. These results suggest that some inefficient DsbA oxidation pathways, such as direct air oxidation, become apparent in the absence of protein synthesis, such that DsbA becomes oxidized in the absence of the functional respiratory chain or even in the absence of DsbB. However, in normally growing cells with actively ongoing protein synthesis and protein export, the functional respiratory electron transfer chain is required for the efficient reoxidation of DsbA.

Figure 5.

Reoxidation of DsbA after inhibition of protein synthesis. Strains H500 (ΔhemA; A and B) and SS141 (dsbB::kan5; C) were grown for two cycles in the absence of ALA as described in Fig. 2A, and H₂O (A) or chloramphenicol (B and C) was added. Samples were withdrawn at 0 (lane 1), 10 (lane 2), 20 (lane 3), and 40 (lane 4) min for determination of redox states of DsbA.

DsbB Is Reduced and Converted to a Disulfide Complex with DsbA After Deprivation of Ubiquinone and Menaquinone.

The results so far presented concerned with the redox state of DsbA. It is important to address whether DsbB is also affected by a lack of the respiratory chain components. We prepared antiserum against a synthetic peptide of carboxyl-terminal 14-amino acid sequence of DsbB. An affinity-purified antibodies decorated a band at an apparent molecular mass of 23 kDa after AMS modification (Fig. 6A, lane 3) or of ≈21 kDa after iodoacetamide modification (Fig. 6C). Note that DsbB contains a total of six cysteines, among which four are essential (37) and presumably disulfide-bonded (20), while the two nonessential cysteines might be in sulfhydryl state and subject to modification with AMS. The intensity of this band increased in cells harboring a dsbB plasmid (Fig. 6A, lane 2), while it was undetectable with the dsbB-disrupted mutant cells (Fig. 6A, lane 1). Thus, we were able to detect the DsbB protein in the cell. Previously, only overproduced and tagged derivatives of DsbB had been reported (18–20, 37).

Figure 6.

Effects of ubiquinone and menaquinone deprivation on intracellular states of DsbB. Cells of TA58 (dsbB::cat; lane 1), CU141/pSS39 (DsbB-overproducer; lane 2), AN387 (ubi⁺ men⁺; lane 3), and AN384 (ubiA420 menA401; lanes 4–8) were grown in L-glucose medium supplemented with potassium phosphate buffer and PHB. Isopropyl β-d-thiogalactoside (1 mM) was added to the culture for lane 2. The cultures for lanes 4–8 were washed with the same medium without PHB, and grown in the PHB-free medium for 1, 2, 3, 4, and 5 hr, respectively. Samples were treated with AMS (A and B), or with iodoacetamide (C), electrophoresed, and subjected to Western blot analysis using affinity-purified anti-DsbB antibodies (A and C) or anti-DsbA serum (B). Although two bands are seen in the region where DsbA–DsbB was electrophoresed in B, only the lower band responded to quinone deprivation in other experiments, indicating that the upper band was a background. Samples for C were treated with 1.4 M 2-mercaptoethanol before electrophoresis. For electrophoresis, samples corresponding to about 10⁷, 4 × 10⁷, and 1.5 × 10⁷ cells were used for A, B, and C, respectively.

The ubiA-menA mutant cells were grown in the absence of PHB, and AMS-treated cell extracts were examined. It was found that DsbB of normal electrophoretic mobility disappeared when the mutant cells were grown in the absence of PHB for 4 hr or longer (Fig. 6A, lanes 7 and 8). After 2 hr, a band of slightly slower than normal migration (at a position of about 25 kDa) appeared (Fig. 6A, lane 5). Presumably, this band represented a reduced form of DsbB. At 3 hr after removal of PHB a band of apparent molecular mass of 43 kDa appeared and continued to exist thereafter (Fig. 6A, lanes 6–8, see the band marked DsbA–DsbB). When the extracts were treated with iodoacetamide and electrophoresed under non-reducing conditions, a higher molecular mass band was also observed at a position of about 41 kDa, which disappeared after reduction with 2-mercaptoethanol (data not shown). DsbB of normal electrophoretic mobility was observed for all the samples when they had been treated with a reducing agent (Fig. 6C). These results show that DsbB is converted to a complex with some intermolecular disulfide linkage after deprivation of quinones. It was also shown that the deprivation did not simply deplete DsbB (Fig. 6C), excluding the possibility that the respiration deficiency somehow shuts off the expression of the dsbB gene.

The same extracts as examined in lanes 4–8 of Fig. 6A were also examined for intracellular states of DsbA (Fig. 6B). It was shown that reduced DsbA started to accumulate at 4 hr. It was also noticed that a band of the same mobility as the higher molecular mass band was also decorated by the DsbA antibodies (Fig. 6B, lanes 6–8).

The electrophoretic mobility of the complex was similar to that of the complex formed between the Cys-33-less mutant of DsbA and DsbB (18, 19). Results of Western blot analysis with anti-DsbA serum are consistent with the notion that the complex contained both DsbB and DsbA. To demonstrate the formation of a DsbB-DsbA disulfide complex under the quinone deprived conditions more conclusively, iodoacetamide-treated extracts were subjected to nonreducing/reducing two-dimensional gel electrophoresis. Staining with anti-DsbB revealed a spot off the diagonal line, whose position in the first dimension was identical to that of the high molecular mass complex and whose second dimension mobility was identical to that of DsbB (Fig. 7, panel 4, arrow). Another spot of the same first dimension mobility and of a DsbA-like second dimension mobility was stained with anti-DsbA (Fig. 7, panel 2). No corresponding spots were detected in wild-type cells (Fig. 7, panels 1 and 3). From these results, taken together, we conclude that DsbB forms a complex with DsbA in the absence of respiratory components.

Figure 7.

Liberation of DsbA and DsbB upon reduction of the higher molecular mass complex. AN387 (ubi⁺ men⁺; panels 1 and 3) and AN384 (ubiA420 menA401; panels 2 and 4) cells were grown in the PHB-free medium for 10 hr (with appropriate dilutions), and iodoacetamide-treated samples were prepared. Samples were subjected to SDS/PAGE without any reducing agent (first dimension). The gel lanes were then cut out, incubated in SDS/sample buffer containing 1.4 M 2-mercaptoethanol at room temperature for 30 min, and layered on the top of the second dimension gel. After electrophoresis, gels were subjected to Western blot analysis using anti-DsbA serum (panels 1 and 2) or anti-DsbB antibodies (panels 3 and 4). Arrows indicate spots of DsbA (panel 2) and DsbB (panel 4).

The results in Fig. 6 indicate the following order of events that occurred after onset of quinone deprivation. First, DsbB is reduced and then it is converted to a DsbA–DsbB complex. The disappearance of the normal DsbB is followed by reduction of a bulk of DsbA in the cell. Thus, respiratory function may act primarily on DsbB.

DISCUSSION

In this work, we examined in vivo redox states of DsbA using an improved method, which allows unequivocal separation of the reduced and oxidized forms of this protein by electrophoresis. In addition, the direct trichloroacetic acid treatment minimized artifacts that can be encountered in examinations of redox-active proteins like DsbA (16).

We now demonstrated that deprivation of components of the respiratory chain causes dramatic accumulation of reduced DsbA molecules in the cell. Since this phenotype of the hemA mutant, expressed in the absence of ALA in the medium, could be reversed by addition of hemin chloride, deprivation of protoheme was responsible for the defect. Thus, involvement of (a) cytochrome(s) is strongly suggested. Similarly, involvement of ubiquinone was suggested by the striking accumulation of the reduced form of DsbA when the mutationally altered ubiA gene product was rendered inactive. Since the ubiA single mutant accumulated only a small proportion of reduced DsbA (T.K., S.K. and K.I., unpublished results), menaquinone is also involved in the reoxidation mechanism of DsbA. A simplest interpretation of these results seems to be that the functional electron transfer chain is required for the efficient oxidation of the DsbA–DsbB system.

Our results show that cells accumulate reduced form of DsbA only when they are growing under the conditions of limited availability of the respiratory components. Acidification of the medium must be avoided to allow continuous growth of the respiratory mutants on glucose fermentation. Even under these conditions we always detected a small proportion of oxidized DsbA. Inhibition of protein synthesis by chloramphenicol resulted in oxidation of DsbA. We assume that the oxidation of DsbA observed at a stationary growth phase is also due to a lack of active protein synthesis. Under such conditions, DsbA will meet few newly synthesized secretory proteins that can be its substrates. A decreased supply of substrate proteins means a decreased probability of reduction of DsbA. Thus, the requirement for the respiratory chain in the maintenance of the oxidized state of DsbA was alleviated in the absence of active protein synthesis. Even the dsbB-disrupted mutant produced some oxidized DsbA in the presence of chloramphenicol. These results suggest that there are background pathways of DsbA oxidation, including a DsbB-independent one. However, the respiratory chain-DsbB coupling must work to keep DsbA oxidized, coping with active synthesis of secretory proteins.

Earlier observations indicated that flagellum synthesis requires the intact electron transfer system (38, 39). This can be now explained by a disulfide bond formation defect, as it has been established that biogenesis of flagellum absolutely depends on the Dsb system (40). Our results showed that a large fraction of β-lactamase accumulated as the reduced form in the heme-deprived cells.

If respiratory chain is required for the DsbA–DsbB system, its primary target might be DsbB, which then acts on DsbA. According to the model we proposed (20), a more specific target of the respiration-dependent oxidation might be the thioredoxin-like motif, Cys-41-Val-Leu-Cys-44, in the amino-terminal periplasmic domain of DsbB. We found that deprivation of quinones had drastic effects on the intracellular states of DsbB. This protein was converted into a DsbB-DsbA complex that was held together by an intermolecular disulfide bond. While DsbB was almost quantitatively converted into the complex, only a small fraction of DsbA was in the complex. Thus, DsbA should exist in excess over DsbB in the cell.

Upon onset of ubiquinone deprivation, the transient reduction of DsbB and its conversion into the DsbA–DsbB complex preceded the reduction of the bulk of DsbA (Fig. 6). These results show that the respiration deficiency first affect the cellular states of DsbB, which was then followed by accumulation of the reduced form of DsbA. Thus, respiratory chain-dependent oxidation system acts primarily on DsbB.

Why then does DsbB accumulate in a form of the DsbA–DsbB complex? It was demonstrated previously (19, 20) that a DsbA mutant lacking the Cys-33 residue forms a disulfide complex with DsbB, in which the Cys-30 of DsbA and Cys-104 of DsbB are disulfide bonded. It was interpreted that this complex represented an intermediate in the DsbB-dependent DsbA oxidation reaction; because Cys-33, the partner of Cys-30, was lacking, the intermediate could not be resolved to the final products. It should be noted that we observed that the mutant form of DsbA formed disulfide-linked products with a number of other cellular proteins as well, when cells were grown in the presence of a disulfide compound such as glutathione disulfide. However, in the absence of glutathione disulfide, it specifically forms the DsbA–DsbB complex, suggesting that DsbA has high affinity to DsbB (18).

We propose the following working model to explain the experimental results obtained in this work. In the absence of respiration, a DsbA–DsbB complex is formed presumably between the same combination of cysteine residues, Cys-30 of DsbA and Cys-104 of DsbB. This complex represents either an intermediate state in normal recycling of the system or a fortuitous product due to a high affinity between DsbA and DsbB as well as the high reactivity of the cysteine residues. In any case, the complex formation is probably unidirectional and its resolution by reverse reaction is somehow prevented. Instead, it is only resolved by the attack of the amino-terminally located Cys-41–Cys-44 disulfide bond of DsbB. This reaction can either be intramolecular, as proposed previously (20), or intermolecular. The lack of the respiratory function may lead to the failure of effective oxidation of the Cys-41 and Cys-44 residues, which in turn leads to the failure in resolution of the DsbA–DsbB disulfide complex. We indeed observed a transient form of DsbB, which was most probably in a reduced state (Fig. 6A, lanes 5 and 6). Although many aspects of this model need further clarification and verification, it at least gives some guidance for the next experimental approaches to the problem.

It is crucial to identify the factor that directly interacts with DsbB and accept electrons. Such a factor may also be interacting with a respiratory chain component. Whether this oxidation involves disulfide-sulfhydryl exchange reaction will be a major question to be addressed. It is interesting to note that many components of electron transfer chains require the Dsb system for their biogenesis, and that this requirement might include processes other than disulfide bond formation (41–43).

The present study opens up new research directions in the fields of cellular energy metabolism and assisted protein folding. Elucidation on the mechanisms of oxidation of DsbA/DsbB will unravel the previously unrecognized physiological roles of the electron transfer system in oxidative protein folding.

ABBREVIATIONS

AMS

4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate

PHB

p-hyroxybenzoate

ALA

5-aminolevulinic acid