Mixed Disulfide Intermediate Research Articles

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

Functional Analysis and Characterization of the Metal Bound Sco Protein from Thermus thermophilus

Cytochrome c oxidase (CcO) is the final complex of the respiratory electron transport chain, which is a set of complexes that participate in the production of adenosine triphosphate (ATP), the main form of energy used by cells. CuA is the initial electron acceptor of CcO and consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine and the carbonyl backbone of a nearby glutamine. Sco proteins have been implicated in the assembly of the CuA, site but the exact role that it plays is the subject of much debate and study. Previous work has found that Sco proteins share a fold similar to thioredoxins, pointing toward the possibility of thiol‐disulfide oxidoreductase activity as a function of the protein. The ability of Thermus thermophilus Sco (TtSco) to act as a thiol‐disulfide oxidoreductase has been probed using an experiment to trap the protein‐protein intermediate between TtSco and its target, Thermus thermophilus CuA (TtCuA). The effect of nearby amino acids and metal binding prior to reaction have been determined for the formation of the protein‐protein intermediate, the mixed disulfide intermediate (MDI). The formation of MDI was monitored with either UV‐Visible spectroscopy by observing an absorbance increase at 412 nm as a chromophore tag, TNB, was released upon reaction between TtSco and TtCuA or by non‐reducing SDS‐PAGE gels. Altering Arg 48, which is adjacent to the attacking cysteine to an Asp has a very large effect on MDI production. TtSco also has metal binding ability, found to bind Cu(I), Cu(II), Ni(II), Cd(II) and Co(II) with varying relative affinities, in addition to disulfide reductase activity. To further characterize MDI formation with TtSco, the impact of conserved residues involved in metal binding on MDI formation were probed and showed that changing the conserved histidine did not affect MDI formation. However, binding of metal to Sco prior to incubating with TtCuA abrogated MDI formation. In characterizing the metal bound protein, preliminary studies to determine the binding affinity of Cu(II) to TtSco have been completed. The impact of conserved cysteines on metal binding has also been studied and the results indicate that one cysteine binds much more tightly to the Cu(II) than the other. While this work allows for a better understanding of TtSco function, much work remains to be done to understand the impacts of metal binding and to further characterize the Sco family of proteins.Support or Funding InformationResearch Corporation (Cottrell College Science Award 7963), the Welch Foundation (W‐0031 [Trinity Chemistry Department]), FASTER grant SURF‐ National Science Foundation DUE S‐STEM Award 1153796 and the Arnold and Mabel Beckman Foundation Beckman Scholars AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate.

The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the CuA protein from T. thermophilus, which is a soluble protein engineered from subunit II of cytochrome ba 3 oxidase that lacks the transmembrane helix. The native cysteines on TtSco and TtCuA were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in TtCuA and subsequent release upon incubation with the complementary TtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine of TtSco that attacks the disulfide bond in the target TtCuA protein was determined to be TtSco Cysteine 49. This cysteine is likely more reactive than Cysteine 53 due to a higher degree of solvent exposure. Removal of the metal binding histidine, His 139, does not change MDI formation. However, altering the arginine adjacent to the reactive cysteine in Sco (Arginine 48) does alter the formation of the MDI. Binding of Cu2+ or Cu+ to TtSco prior to reaction with TtCuA was found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by the TtSco protein and may point to a possible role of metal binding in regulating the activity. IMPORTANCE: The function of Sco is at the center of many studies. The disulfide bond reduction in CuA by Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.

Structural and Functional Characterization of the Sco Protein from Thermus thermophilus

The Sco family of proteins has been implicated in the assembly of cytochrome c oxidase (Complex IV) of the respiratory electron transport chain, but its function is still under debate. TtSco has been shown to bind to Cu (I) and Cu (II) ions. However, it is also structurally similar to thioredoxins and has been shown to be able to reduce the disulfide bond present in the CuA site of cytochrome c oxidase prior to copper insertion. To provide evidence of Sco's role in the assembly of the CuA‐center, single cysteine to serine mutants, TtSco‐C53S and TtCuA‐C149S, were first produced. TtCuA was conjugated to a chromophore that is released upon reaction with TtSco. The two proteins were reacted and monitored via UV‐Visible spectroscopy in a 1:1 ratio at 412 nm to monitor formation of a mixed disulfide intermediate (MDI) between the two. TtSco and TtCuA have been reacted to form the MDI at different stoichiometric ratios and to probe the kinetics of formation of the MDI. Our results indicate much higher production of MDI with Sco in excess. Our second aim is to crystallize the mixed disulfide intermediate to visualize and better understand the interactions between TtSco and TtCuA. Cation exchange chromatography has shown the most success in separating MDI from unreacted starting materials. A successful condition for crystallization of the MDI has been found and consists of ammonium sulfate and polyethylene glycol 8,000. Lastly, the effects of copper binding on the relative stability of apo and holo forms of TtSco have been studied via chemical denaturation. Interestingly, preliminary data suggests that holo‐TtSco is slightly less stable than apo‐TtSco. Further studies aim to investigate the effect of the copper‐ligating His139 on stability of the protein, both with and without metal bound to gain more insight into TtSco's ability to bind metals and the role metal binding plays in its function.Support or Funding InformationWelch Foundation

Reversible S-nitrosylation limits over synthesis of fungal styrylpyrone upon nitric oxide burst.

Nitric oxide (NO) is known to be involved in modulating production of styrylpyrone polyphenols in the basidiomycete Inonotus obliquus. However, it remains unknown how NO orchestrates fungal styrylpyrone biosynthesis. Here, we show that a transient NO burst correlated with an enhanced expression of phenylalanine ammonia lyase (PAL), 4-coumarate CoA ligase (4CL), and styrylpyrone synthase (SPS), the key enzymes involved in styrylpyrone biosynthesis, and subsequently an increased production of styrylpyrone polyphenols. In parallel, the NO burst also resulted in S-nitrosylation of PAL, 4CL, and SPS, which compromised their enzymatic activities mediating a post-translational feedback mechanism that keeps NO-dependent transcriptional activation in check. Moreover, dysfunction of thioredoxin reductase (TrxR) further increased the formation of S-nitrosylated proteins, implicating the significance of the Trx system in maintaining a low level of protein-nitrosothiols. Three thioredoxin-like proteins (TrxLs) from I. obliquus show in vitro denitrosylation potential toward S-nitrosylated proteins via trans-denitrosylation or mixed disulfide intermediates. Thus, S-nitrosylation triggered by the NO burst limits over production of fungal styrylpyrone polyphenols, and denitrosylation by TrxLs that act in concert with TrxR play a key role in maintaining redox balance and orchestrating catalytic activities of the enzymes engaged in styrylpyrone synthetic metabolism.

Redox biology: signaling via a peroxiredoxin sensor.

Hydrogen peroxide regulates cell signaling pathways through oxidation of specific thiol proteins. A new study describes a relay system involving peroxiredoxin 2 as a peroxide sensor that oxidizes the mammalian transcription factor STAT3 via a mixed disulfide intermediate.

How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation*♦

Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

Mia40 is optimized for function in mitochondrial oxidative protein folding and import.

Mia40 catalyzes oxidative protein folding in mitochondria. It contains a unique catalytic CPC dithiol flanked by a hydrophobic groove, and unlike other oxidoreductases, it forms long-lived mixed disulfides with substrates. We show that this distinctive property originates neither from particular properties of mitochondrial substrates nor from the CPC motif of Mia40. The catalytic cysteines of Mia40 display unusually low chemical reactivity, as expressed in conventional pK values and reduction potentials. The stability of the mixed disulfide intermediate is coupled energetically with hydrophobic interactions between Mia40 and the substrate. Based on these properties, we suggest a mechanism for Mia40, where the hydrophobic binding site is employed to select a substrate thiol for forming the initial mixed disulfide. Its long lifetime is used to retain partially folded proteins in the mitochondria and to direct folding toward forming the native disulfide bonds.

Placeholder Mechanism of Catalyzed Oxidative Folding is Conserved Across Multiple Domains of Life

Disulfide oxidoreductases react with substrates through a mixed disulfide intermediate. A second cysteine from either substrate or enzyme can attack the mixed disulfide bond, resulting in substrate oxidation or enzyme release, respectively. During catalyzed oxidative folding, the enzyme acts as a passive placeholder; remaining in a mixed disulfide with a substrate cysteine until folding brings the cognate cysteine into proximity. Therefore, in order for catalyzed oxidative folding to succeed, the enzyme must release with a rate slower than the folding rate of the substrate. We have developed single molecule AFM techniques to monitor the formation of mixed disulfide complexes between substrate and oxidoreductase, which in turn can be used to measure the rates of catalyzed oxidative folding as well as enzyme release. Previously, our group has used these techniques to study PDI, the primary eukaryotic disulfide oxidant. Here we investigate the bacterial analog of PDI, DsbA. For both enzymes, we found that the rate of catalyzed oxidative folding is comparable to the rate of folding of reduced substrate, while a previously oxidized substrate folds roughly 30 times faster. This result entails that oxidation must occur late in the folding process, thus confirming the passive placeholder mechanism. The fact that both enzymes follow the same mechanism suggests that it might be a general model for disulfide oxidoreductase function across all domains of life. Despite the similar mechanisms and in vivo roles of the two enzymes, we found vastly different rates of spontaneous release. DsbA releases at a rate of about 0.01 s-1, which is roughly tenfold slower than PDI. This exceedingly slow rate of release suggests that the repertoire of prokaryotic proteins that are catalytically oxidized may take longer to fold, on average, than their eukaryotic counterparts.

Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells

Hydrogen peroxide is widely viewed as the main second messenger in redox signaling, and it has been proposed that deactivation of the antioxidant peroxiredoxin (Prdx) enzymes allows free peroxide to accumulate and directly oxidize target proteins (the floodgate model). We assessed the role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway, in which oxidation of ASK1 is required for phosphorylation of p38. In response to peroxide, Prdx1 catalyzed oxidation of ASK1 to a disulfide-linked multimer, and this occurred via transient formation of a Prdx1—ASK1 mixed disulfide intermediate. Oxidation of ASK1 and phosphorylation of p38 were inhibited by knockdown of Prdx1, but also by overexpression of Prdx2. This suggests that these two cytosolic Prdxs have distinct roles in the cellular peroxide response and compete for available peroxide substrate. These data imply that Prdx1 can function as a peroxide receptor in response to extracellular H2O2, receiving the peroxide signal and transducing it into a disulfide bond that is subsequently transmitted to the substrate, ASK1, resulting in p38 phosphorylation. Interestingly, in response to peroxide, Prdx1 and Prdx3 transiently formed reducible higher molecular weight complexes, suggesting that multiple proteins are targets for Prdx-mediated oxidation via a disulfide-exchange mechanism. This model of active peroxide signal distribution via disulfide exchange is consistent with Prdx function in yeast and explains how peroxides may trigger specific disulfide bond formation in mammalian cells.

The N-terminal Shuttle Domain of Erv1 Determines the Affinity for Mia40 and Mediates Electron Transfer to the Catalytic Erv1 Core in Yeast Mitochondria

Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.

Structural Basis for the Disulfide Relay System in the Mitochondrial Intermembrane Space

Mitochondria contain two biological membranes. Although reducing agents can diffuse from the cytosol into the intermembrane space (IMS) between the outer and inner mitochondrial membranes, the IMS has a dedicated disulfide relay system to introduce disulfide bonds into mainly small and soluble proteins. This system consists of two essential proteins, a disulfide carrier Tim40/Mia40 and a flavin-dependent sulfhydryl oxidase Erv1, high-resolution structures that have recently become available. Tim40/Mia40 transfers disulfide bonds to newly imported IMS proteins by dithiol/disulfide exchange reactions involving mixed disulfide intermediates. Tight folding by introduction of disulfide bonds prevents egress of these small IMS proteins, resulting in their selective retention in the compartment. After disulfide transfer from Tim40/Mia40 to substrate proteins, Tim40/Mia40 is reoxidized again by Erv1, which is then oxidized by electron transfer to either cytochrome c or molecular oxygen. Here we review the recent advancement of the knowledge on the mechanism of the disulfide relay system in the mitochondrial IMS, especially shedding light on the structural aspects of its components.

Target proteins of the cytosolic thioredoxin in Plasmodium falciparum

The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and l-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.

Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Redox Receptor Regulation Revealed

Thioredoxin-1 (Trx1) is a thiol-disulfide oxidoreductase that can reduce transiently formed disulfide bonds in substrate proteins. The enzyme is released from lymphocytes and has extracellular cytokine-like actions, but the mechanisms by which it produces such effects are not known. Schwertassek et al . used a modified form of Trx1 in a substrate trapping assay (in which the engineered Trx1 formed long-lived mixed disulfide intermediates with its targets) to search for cell-surface proteins that might be modified by the enzyme. An affinity tag on the modified Trx1 allowed purification of the complexes and subsequent identification of associated proteins by mass spectrometry. In the human Epstein-Barr virus--transformed lymphoblastoid B cell line LCL-721.220--the authors found just one primary target of Trx-1, a member of the tumor necrosis factor receptor (TNFR) superfamily known as TNFRSF8 or CD30. The association of the proteins was lost under reducing conditions and, although all TNFR superfamily members characteristically have cysteine-rich domains, association with Trx1 was specific to CD30. Modification of CD30 appeared to cause a conformational change in the receptor because recognition of the receptor by antibodies was altered. This action of Trx1 required the presence of a source of reducing equivalents. Incubation of Hodgkin’s lymphoma HDLM-2 cells decreased binding of recombinant soluble CD30 ligand to its receptor at the cell surface and, in the YT granular lymphoma cell line, blocked increased expression of the gene encoding the interleukin-2 receptor alpha chain, which is normally activated by CD30 signaling. The authors thus propose that excreted Trx1 may alter receptor function and may, in this way, link oxidative stress to lymphocyte function. U. Schwertassek, Y. Balmer, M. Gutscher, L. Weingarten, M. Preuss, J. Engelhard, M. Winkler, T. P. Dick, Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1. EMBO J. 26 , 3086-3097 (2007). [PubMed]

Structures of Ternary Complexes of BphK, a Bacterial Glutathione S-Transferase That Reductively Dechlorinates Polychlorinated Biphenyl Metabolites

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic structures of two ternary complexes of BphK were solved to a resolution of 2.1A. In the BphK-GSH-HOPDA complex, GSH and HOPDA molecules occupy the G- and H-subsites, respectively. The thiol nucleophile of the GSH molecule is positioned for SN2 attack at carbon 3 of the bound HOPDA. The respective sulfur atoms of conserved Cys-10 and the bound GSH are within 3.0A, consistent with product release and the formation of a mixed disulfide intermediate. In the BphK-(GSH)2 complex, a GSH molecule occupies each of the two subsites. The three sulfur atoms of the two GSH molecules and Cys-10 are aligned suitably for a disulfide exchange reaction that would regenerate the resting enzyme and yield disulfide-linked GSH molecules. A second conserved residue, His-106, is adjacent to the thiols of Cys-10 and the GSH bound to the G-subsite and thus may stabilize a transition state in the disulfide exchange reaction. Overall, the structures support and elaborate a proposed dehalogenation mechanism for BphK and provide insight into the plasticity of the H-subsite.

The N-terminal Domain of PILB from Neisseria meningitidis Is a Disulfide Reductase That Can Recycle Methionine Sulfoxide Reductases

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.

7] Disulfide bond formation in periplasm of Escherichia coli

Publisher Summary This chapter outlines the process of disulfide bond formation in periplasm of Escherichia coli . The formation of stable disulfide bonds in proteins takes place in specific compartments in the cell. The periplasm of Escherichia coli contains a mechanism devoted to the correct and rapid formation of disulfide bonds. This machinery comprises two main thiol/disulfide exchange processes: (1) the enzymatic oxidation of free protein thiols into disulfide bonds and (2) the disulfide bond isomerization or reshuffling of incorrect disulfide bonds. This chapter summarizes the genetic approaches that have been used to identify the components of this machinery. In addition, it describes protocols for detecting and quantitating these two E. coli periplasmic thiol/disulfide activities in vivo. Finally, the chapter describes a technique for detecting mixed disulfide intermediates formed in vivo by the rapid reactions carried out by thiol/disulfide oxidoreductases. This technique allows the definition of aspects of the reaction mechanism and the identification of novel substrates of these enzymes.

The Role of the Buried Aspartate of Escherichia coliThioredoxin in the Activation of the Mixed Disulfide Intermediate

The structurally homologous protein disulfide isomerases and thioredoxins exhibit a 10(5) variation of redox equilibria. It is demonstrated that the kinetic distinction among these protein family members lies primarily in the rate of breakdown of the mixed disulfide intermediate. The conserved buried acid group serves as a proton transfer catalyst for the buried active site cysteine in the formation and breakdown of the mixed disulfide. The reduction rate of Escherichia coli thioredoxin by dithiothreitol is directly proportional to the fraction of Asp-26 in the protonated form over the pH range of 6-9. The kinetic role of Asp-26 is further probed via differential solvent kinetic isotope effect measurements versus a D26N variant. The differential solvent isotope effect of 0.6 is consistent with a direct proton donation to the thiolate leaving group (Cys-35) via an enforced general acid catalysis by trapping mechanism. Such a donation necessitates a structural rearrangement as these two buried side chains are separated by 6 A in both the oxidized and reduced forms of the protein.

Reduction of protein disulfide bonds in an oxidizing environment: The disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum

Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge [Majoul et al. (1996) J. Cell Biol. 133, 777–789], although the redox state in the ER favors disulfide formation. We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3. Protein disulfide isomerase (PDI) exerts only a minor accelerating effect. Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction. These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.