Disulfide Bond Formation Pathway Research Articles

Effects of Amylopectins from Five Different Sources on Disulfide Bond Formation in Alkali-Soluble Glutenin.

Wheat, maize, cassava, mung bean and sweet potato starches have often been added to dough systems to improve their hardness. However, inconsistent effects of these starches on the dough quality have been reported, especially in refrigerated dough. The disulfide bond contents of alkali-soluble glutenin (ASG) have direct effects on the hardness of dough. In this paper, the disulfide bond contents of ASG were determined. ASG was mixed and retrograded with five kinds of amylopectins from the above-mentioned botanical sources, and a possible pathway of disulfide bond formation in ASGs by amylopectin addition was proposed through molecular weight, chain length distribution, FT-IR, 13C solid-state NMR and XRD analyses. The results showed that when wheat, maize, cassava, mung bean and sweet potato amylopectins were mixed with ASG, the disulfide bond contents of alkali-soluble glutenin increased from 0.04 to 0.31, 0.24, 0.08, 0.18 and 0.29 μmol/g, respectively. However, after cold storage, they changed to 0.55, 0.16, 0.26, 0.07 and 0.19 μmol/g, respectively. The addition of wheat amylopectin promoted the most significant disulfide bond formation of ASG. Hydroxyproline only existed in the wheat amylopectin, indicating that it had an important effect on the disulfide bond formation of ASG. Glutathione disulfides were present, as mung bean and sweet potato amylopectin were mixed with ASG, and they were reduced during cold storage. Positive/negative correlations between the peak intensity of the angles at 2θ = 20°/23° and the disulfide bond contents of ASG existed. The high content of hydroxyproline could be used as a marker for breeding high-quality wheat.

Characterization of the endoplasmic reticulum–resident peroxidases GPx7 and GPx8 shows the higher oxidative activity of GPx7 and its linkage to oxidative protein folding

Oxidative protein folding occurs primarily in the mammalian endoplasmic reticulum, enabled by a diverse network comprising more than 20 members of the protein disulfide isomerase (PDI) family and more than five PDI oxidases. Although the canonical disulfide bond formation pathway involving Ero1α and PDI has been well-studied so far, the physiological roles of the newly identified PDI oxidases, glutathione peroxidase-7 (GPx7) and -8 (GPx8), are only poorly understood. We here demonstrated that human GPx7 has much higher reactivity with H2O2 and hence greater PDI oxidation activity than human GPx8. The high reactivity of GPx7 is due to the presence of a catalytic tetrad at the redox-active site, which stabilizes the sulfenylated species generated upon the reaction with H2O2 Although it was previously postulated that GPx7 catalysis involved a highly reactive peroxidatic cysteine that can be sulfenylated by H2O2, we revealed that a resolving cysteine instead regulates the PDI oxidation activity of GPx7. We also determined that GPx7 formed complexes preferentially with PDI and P5 in H2O2-treated cells. Altogether, these results suggest that human GPx7 functions as an H2O2-dependent PDI oxidase in cells, whereas PDI oxidation may not be the central physiological role of human GPx8.

Per Os Infectivity Factor 5 Identified as a Substrate of P33 in the Baculoviral Disulfide Bond Formation Pathway.

Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection.IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulfide-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.

Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation.

Disulfide bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coliIMPORTANCE Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.

The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli.

Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited.IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

Inhibition of virulence-promoting disulfide bond formation enzyme DsbB is blocked by mutating residues in two distinct regions

Disulfide bonds contribute to protein stability, activity, and folding in a variety of proteins, including many involved in bacterial virulence such as toxins, adhesins, flagella, and pili, among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In the Escherichia coli disulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates, and then the cytoplasmic membrane protein DsbB reoxidizes DsbA's cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bond de novo by transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several Gram-negative bacteria. To map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR the E. coli dsbB gene and selected dsbB mutants that confer resistance to this drug using two approaches. We characterized in vivo and in vitro some of these mutants that map to two areas in the structure of DsbB, one located between the two first transmembrane segments where the quinone ring binds and the other located in the second periplasmic loop of DsbB, which interacts with DsbA. In addition, we show that a mutant version of a protein involved in lipopolysaccharide assembly, lptD4213, is synthetically lethal with the deletion of dsbB as well as with DsbB inhibitors. This finding suggests that drugs decreasing LptD assembly may be synthetically lethal with inhibitors of the Dsb pathway, potentiating the antibiotic effects.

Identification and characterization of GmPDIL7, a soybean ER membrane-bound protein disulfide isomerase family protein.

The nucleotide sequence data for the GmPDIL7 cDNA are available in the DNA Data Bank of Japan (DDBJ) databases under the accession numbers LC158001. Protein disulfide isomerase: EC 5.3.4.1.

Maternal nicotine exposure leads to impaired disulfide bond formation and augmented endoplasmic reticulum stress in the rat placenta.

Maternal nicotine exposure has been associated with many adverse fetal and placental outcomes. Although underlying mechanisms remain elusive, recent studies have identified that augmented endoplasmic reticulum (ER) stress is linked to placental insufficiency. Moreover, ER function depends on proper disulfide bond formation—a partially oxygen-dependent process mediated by protein disulfide isomerase (PDI) and ER oxidoreductases. Given that nicotine compromised placental development in the rat, and placental insufficiency has been associated with poor disulfide bond formation and ER stress, we hypothesized that maternal nicotine exposure leads to both placental ER stress and impaired disulfide bond formation. To test this hypothesis, female Wistar rats received daily subcutaneous injections of either saline (vehicle) or nicotine bitartrate (1 mg/kg) for 14 days prior to mating and during pregnancy. Placentas were harvested on embryonic day 15 for analysis. Protein and mRNA expression of markers involved in ER stress (e.g., phosphorylated eIF2α, Grp78, Atf4, and CHOP), disulfide bond formation (e.g., PDI, QSOX1, VKORC1), hypoxia (Hif1α), and amino acid deprivation (GCN2) were quantified via Western blot and/or Real-time PCR. Maternal nicotine exposure led to increased expression of Grp78, phosphorylated eIF2α, Atf4, and CHOP (p<0.05) in the rat placenta, demonstrating the presence of augmented ER stress. Decreased expression of PDI and QSOX1 (p<0.05) reveal an impaired disulfide bond formation pathway, which may underlie nicotine-induced ER stress. Finally, elevated expression of Hif1α and GCN2 (p<0.05) indicate hypoxia and amino acid deprivation in nicotine-exposed placentas, respectively, which may also cause impaired disulfide bond formation and augmented ER stress. This study is the first to link maternal nicotine exposure with both placental ER stress and disulfide bond impairment in vivo, providing novel insight into the mechanisms underlying nicotine exposure during pregnancy on placental health.

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30% higher enzyme yields (16,200U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space-time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.

Structural and Functional Characterization of ScsC, a Periplasmic Thioredoxin-Like Protein fromSalmonella entericaSerovar Typhimurium

The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of β-sheets and α-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity.

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40-substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40-substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.

Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals

Almost all organisms, from bacteria to humans, possess catalytic systems that promote disulfide bond formation-coupled protein folding, i.e. oxidative protein folding. These systems are necessary for the biosynthesis of many secretory and membrane proteins, such as antibodies, major histocompatibility complex molecules, growth factors, and insulin. Over the last decade, structural studies have made striking progress in this field of research, identifying how oxidative systems operate in a specific and regulated manner to maintain redox and protein homeostasis within cells. Interestingly, more and more novel catalysts that promote disulfide bond formation have been discovered in mammals, suggesting that the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought. This review highlights the physiological roles and molecular bases of the disulfide bond formation pathways that have evolved in the bacterial periplasm and the endoplasmic reticulum of fungi and mammals. Accumulating knowledge about disulfide bond formation networks widely distributed throughout the biological kingdom has significantly advanced our understanding of the cellular mechanisms dedicated to protein quality control.

The interplay between the disulfide bond formation pathway and cytochrome c maturation in Escherichia coli

Heme attachment to c-type cytochromes in bacteria requires cysteine thiols in the CXXCH motif of the protein. The involvement of the periplasmic disulfide generation system in this process remains unclear. We undertake a systematic evaluation of the role of DsbA and DsbD in cytochrome c biogenesis in Escherichia coli and show unequivocally that DsbA is not essential for holocytochrome production under aerobic or anaerobic conditions. We also prove that DsbD is important but not essential for maturation of c-type cytochromes. We discuss the findings in the context of a model in which heme attachment to, and oxidation of, the apocytochrome are competing processes.

Hunting for Alternative Disulfide Bond Formation Pathways: Endoplasmic Reticulum Janitor Turns Professor and Teaches a Lesson

In this issue of Molecular Cell, Ron and colleagues (Zito et al., 2010b) show that an enzyme responsible for cleaning up hydrogen peroxide in the endoplasmic reticulum can contribute productively to disulfide bond formation.

Cytosolic Disulfide Bond Formation in Cells Infected with Large Nucleocytoplasmic DNA Viruses

Proteins that have evolved to contain stabilizing disulfide bonds generally fold in a membrane-delimited compartment in the cell [i.e., the endoplasmic reticulum (ER) or the mitochondrial intermembrane space (IMS)]. These compartments contain sulfhydryl oxidase enzymes that catalyze the pairing and oxidation of cysteine residues. In contrast, most proteins in a healthy cytosol are maintained in reduced form through surveillance by NADPH-dependent reductases and the lack of sulfhydryl oxidases. Nevertheless, one of the core functionalities that unify the broad and diverse set of nucleocytoplasmic large DNA viruses (NCLDVs) is the ability to catalyze disulfide formation in the cytosol. The substrates of this activity are proteins that contribute to the assembly, structure, and infectivity of the virions. If the last common ancestor of NCLDVs was present during eukaryogenesis as has been proposed, it is interesting to speculate that viral disulfide bond formation pathways may have predated oxidative protein folding in intracellular organelles.

MBSJ MCC Young Scientist Award 2009 REVIEW: Structural basis of protein disulfide bond generation in the cell

The formation of protein disulfide bonds is an oxidative reaction that is crucial for the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells possess various disulfide oxidoreductases and redox‐active cofactors to accelerate this oxidative reaction in a correct manner. Crystal or solution structures have been solved for some of the oxidoreductases in the past 10 years, leading to remarkable progress in the field of thiol‐based redox cell biology. Consequently, structural and mechanistic similarities in the disulfide bond formation pathways have been uncovered. This review highlights the molecular basis of the elaborate oxidative systems operating in the Escherichia coli periplasm, the endoplasmic reticulum lumen and the mitochondrial intermembrane space. The accumulated knowledge provides important insights into how protein and redox homeostasis are maintained in the cell.

Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation

Protein disulfide bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about disulfide bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for disulfide bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of disulfide bond formation, with data on the presence of homologs of known disulfide bond formation enzymes. These combined data enabled us to make predictions about disulfide bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.

Conservation and Diversity of the Cellular Disulfide Bond Formation Pathways

Two pathways for the formation of biosynthetic protein disulfide bonds have been characterized in the endoplasmic reticulum (ER) of eukaryotes. In the major pathway, the membrane-associated flavoprotein Ero1 generates disulfide bonds for transfer to protein disulfide isomerase (PDI), which is responsible for directly introducing disulfide bonds into secretory proteins. In a minor fungal-specific protein oxidation pathway, the membrane-associated flavoprotein Erv2 can catalyze disulfide bond formation via the transfer of oxidizing equivalents to PDI. Genomic sequencing has revealed an abundance of enzymes sharing homology with Ero1, Erv2, or PDI. Herein the authors discuss the functional, mechanistic, and potential structural similarities between these homologs and the core enzymes of the characterized ER oxidation pathways. In addition they speculate about the possible differences between these enzymes that may explain why the cell contains multiple proteins dedicated to a single process. Finally, the eukaryotic ER protein oxidation and reduction pathways are compared to the corresponding prokaryotic periplasmic pathways, to highlight the functional, mechanistic, and structural similarities that exist between the pathways in these two kingdoms despite very low primary sequence homology between the protein and small molecule components.

Generating disulfides enzymatically: Reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.

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

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 Cys41 and Cys44 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 (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees 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 DsbA-DsbB redox reactions.