Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K_M for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK_a of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK_a values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 Å, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.     

An Extracellular Disulfide Bond Forming Protein (DsbF) from Mycobacterium tuberculosis: Structural, Biochemical, and Gene Expression Analysis

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here, we present structural, biochemical, and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pK_a for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein coexpression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicates anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions.     

Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica

PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

The Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK_a of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK_a of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°_H2O = 9 kJ/mol and ΔT_m = 7. 4 °C) than for Trx1 (ΔΔG°_H2O = 15 kJ/mol and ΔT_m = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn²⁺-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK_a of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn²⁺ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn²⁺-center of Trx2 fine-tunes the properties of this unique thioredoxin.     

Structure of the Catalytic aa Fragment of the Protein Disulfide Isomerase ERp72

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). The ER contains many different PDI-like proteins. Some, such as PDI, are general enzymes that directly recognize misfolded proteins while others, such as ERp57 and ERp72, have more specialized roles. Here, we report the high-resolution X-ray crystal structure of the N-terminal portion of ERp72 (also known as CaBP2 or PDI A4), which contains two a⁰a catalytic thioredoxin-like domains. The structure shows that the a⁰ domain contains an additional N-terminal β-strand and a different conformation of the β5-α4 loop relative to other thioredoxin-like domains. The structure of the a domain reveals that a conserved arginine residue inserts into the hydrophobic core and makes a salt bridge with a conserved glutamate residue in the vicinity of the catalytic site. A structural model of full-length ERp72 shows that all three catalytic sites roughly face each other and positions the adjacent hydrophobic patches that are likely involved in protein substrate binding.     

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.     

Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the Transmembrane Oxidoreductase DsbD

The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys¹⁰³-Cys¹⁰⁹ disulfide of nDsbD by Cys⁴⁶¹ of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. (2007) J. Mol. Biol. 370 ,643 -658) that the attacking cysteine (Cys⁴⁶¹) in isolated cDsbD has a high pK_a value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pK_a values change significantly when cDsbD forms a complex with nDsbD. This modulation of pK_a values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pK_a values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.DsbD is a unique protein that transfers reductant across the cytoplasmic membrane to the periplasm in many Gram-negative bacteria (1, 2). Provision of reductant to the periplasm is required because this compartment is otherwise considered to be an oxidizing environment (2). DsbD includes three domains, each containing a pair of cysteine residues that perform a series of disulfide exchange reactions (Fig. 1A). In the first step, the transmembrane domain (tmDsbD) accepts electrons from thioredoxin in the cytoplasm; these are then transferred to the periplasmic C-terminal domain (cDsbD) and finally to the N-terminal domain (nDsbD), which is also located in the periplasm (3-5). nDsbD acts as a junction point for several pathways that require reductant, including the general disulfide isomerase system and the pathway that is thought to reduce the cysteine thiols of apocytochromes in the cytochrome c biogenesis pathway (6). In Gram-positive bacteria, CcdA, an integral membrane protein, and ResA, which has a thioredoxin fold, provide the reductant required for cytochrome c maturation (7).Open in a separate windowFIGURE 1.Schematic representation of DsbD. A, proposed pathway of electron flow from thioredoxin (TrxA) in the cytoplasm, via the three domains of DsbD, to the cytochrome c maturation (Ccm) and disulfide bond isomerization pathways in the periplasm is shown. The crystal structure of nDsbD is from Protein Data Bank code 1L6P (8), cDsbD from Protein Data Bank code 1UC7 (11), and the nDsbD-cDsbD complex from Protein Data Bank code 1VRS (12). The cyan boxes indicate the thrombin cleavage sites introduced into full-length DsbD to allow detection of the nDsbD-cDsbD complex following its formation in vivo. The cysteine residues are shown in yellow. B, schematic representation of the active site of cDsbD in the covalent complex with nDsbD (12). Some active-site residues of cDsbD are indicated in stick representation and the inter-domain disulfide (Cys⁴⁶¹-SS-Cys¹⁰⁹) is shown in yellow.Structural studies have sought to explain how DsbD functions and interacts with its various partners. The structures of the two soluble periplasmic domains have been determined (Fig. 1A, left). nDsbD has an immunoglobulin-like structure (8, 9) and is the only known thiol:disulfide oxidoreductase with this fold. cDsbD has the more typical thioredoxin fold found in many oxidoreductases; this has the characteristic active-site CXXC motif (10, 11). A covalent complex between single-cysteine variants of each of these two domains was produced in vitro and its x-ray structure solved (12), revealing the interface between the two domains (Fig. 1A, right). Although this mixed disulfide is accepted as a physiological intermediate in the function of DsbD, an in vivo complex between the two soluble domains has not been reported previously (3). Further complexes between nDsbD and its other physiological partners have also been trapped and their structures examined (9, 13). Interestingly, all of the interaction partners of nDsbD are thioredoxin-like proteins; similarities in their folds are congruous with common interaction interfaces (14). However, only cDsbD will reduce nDsbD, whereas nDsbD will reduce several partners. This raises questions about how the direction of reductant flow is maintained and controlled within the series of disulfide-exchange reactions.As part of our structural and mechanistic characterization of DsbD and its domains in solution, we have previously measured by NMR the pK_a values of the active-site cysteine pair, Cys⁴⁶¹ and Cys⁴⁶⁴, of cDsbD (numbered according to the full-length Escherichia coli DsbD sequence) (15). An unusually high pK_a value of 10.5 was measured for the N-terminal cysteine of the CXXC motif, Cys⁴⁶¹, and the pK_a value of the second cysteine, Cys⁴⁶⁴, was significantly higher than the maximum pH value that was studied (pH 12.2). The pK_a value of 10.5 is the highest reported for the N-terminal cysteine of the CXXC motif in a thioredoxin fold. The striking consequence of the elevated pK_a value is that the active-site cysteine of cDsbD, Cys⁴⁶¹, is not strongly nucleophilic, raising critical questions about how this cysteine reacts with the disulfide in nDsbD. It was demonstrated using site-directed mutagenesis that the negatively charged side chains of Asp⁴⁵⁵ and Glu⁴⁶⁸, which are located close to the CXXC motif (Fig. 1B), are responsible for the unusually high pK_a value of Cys⁴⁶¹; mutation of one or both of these residues to Asn and Gln, respectively, resulted in decreases in the pK_a value of Cys⁴⁶¹ from 10.5 to 9.9 (E468Q), to 9.3 (D455N), and to 8.6 (D455N/E468Q). The pK_a values for Asp⁴⁵⁵ were found to be 5.9 and 6.6 in oxidized and reduced cDsbD; these values are significantly higher than the value of ∼4 for an unperturbed aspartic acid. We postulated that the properties of the amino acid side chains in the immediate environment of the cysteines in cDsbD would change upon complex formation with nDsbD, changing the reactivity of the cysteines and explaining how the reaction between the two domains is initiated (15). Specifically, we proposed that an increase in the pK_a value of Asp⁴⁵⁵ upon complex formation would lead to a decrease in the pK_a value of Cys⁴⁶¹, thereby making it a better nucleophile. Stirnimann et al. (10) previously presented pK_a calculations suggesting an increase in the Asp⁴⁵⁵ pK_a value upon complex formation.The aim of this work has been to determine the molecular basis of the control of the reactivity of the active-site cysteine residues in cDsbD, using NMR to compare the active-site properties of cDsbD alone and in its physiological complex with nDsbD. We demonstrate that the pK_a value of Asp⁴⁵⁵ is elevated by at least 1.1 pH units when cDsbD forms a complex with nDsbD. This modulation of the pK_a value is critical for the specificity and function of cDsbD. These in vitro studies are complemented by in vivo studies on complex formation, in which we have trapped the nDsbD-cDsbD complex for the first time. The results of our experiments explain how the intramolecular disulfide cascade within the soluble domains of DsbD functions, and demonstrate the importance of the transmembrane domain in controlling and facilitating complex formation between the soluble domains.     

Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

One of the most popular and simple models for the calculation of pK_as from a protein structure is the semi‐macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK_as. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK_as; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK_as. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK_a values (where the calculation should reproduce the pK_a within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK_a in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK_a should be shifted, and validation of force field parameters for cysteine residues. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase

The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK_a for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK_a between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpK_a; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase.     

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins.

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.     

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Oxidative inactivation of the lymphoid tyrosine phosphatase mediated by both general and active site directed NO donors

Oxidative modification of protein tyrosine phosphatases (PTPs) has recently been recognized as an important regulatory mechanism in biological systems. Reported herein is the oxidative inactivation of the lymphoid tyrosine phosphatase (LYP) with both the general nitrosating reagent sodium nitroprusside (SNP) and also a novel peptide-based nitrosating reagent, Ac-ARLIEDNE(HcyNO)TAREG-NH₂, where HcyNO = S-nitrosohomocysteine. The SNP oxidatively inactivated LYP with a k_inact of 0.383 per min and a K_I of 27.4 μM and mixed-type inactivation kinetics. The peptide was a competitive LYP inactivator with a k_inact of 0.0472 per min and a K_I of 7.00 μM. LYP nitrosation by SNP was characterized by the addition of several NO moieties to the enzyme, while oxidation of LYP by the peptide did not result in the formation of a LYP-NO adduct. We propose that general NO donors promiscuously nitrosate any free cysteine residue while the active-site directed peptide selectively oxidizes the catalytic cysteine residue, resulting in the formation of a disulfide bond between the catalytic cysteine residue and a second cysteine in the active site.     

A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. The function of these cysteines and the significance of their unique location in yeast PDI have been unclear. Previous data (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786) suggest that the internal cysteines exist as a disulfide in the endoplasmic reticulum of Saccharomyces cerevisiae. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.