Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin

The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys(40), the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to < or =4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.     

Biochemical characterization of the thioredoxin domain of Escherichia coli DsbE protein reveals a weak reductant

Thioredoxin (Trx) domain is a typical fold functioning in thiol/disulfide exchange. DsbE protein is one of the Trx-domain containing proteins involved in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble C-terminal Trx domain of DsbE protein was overexpressed and purified to homogeneity. We herein report biochemical characterization of the structural and redox properties of this domain. During redox reaction, the domain undergoes a structural transformation resulting in a more stable reduced form with a free energy difference (DeltaDeltaG(Redox)) of ca. 5 kcal/mol, but the thiol/disulfide exchange exhibits very low reactivity. The standard redox potential (E0') for the active thiol/disulfide is -0.175 V and the pK(a) value of the active cysteine is around 6.8, indicating that the domain acts as a weak reductant. This implies that the membrane-anchored DsbE protein may provide driven reducing power for the redox reaction in the thiol/disulfide exchange pathway.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.     

Influence of the pK(a) value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases

Thioredoxin constitutes the prototype of the thiol-disulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively (13)C(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thiol-disulfide oxidoreductases with reasonable accuracy.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.     

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.     

Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP

Reactivities of the two essential cysteine residues in the heavy metal binding motif, MTC(14)AAC(17), of the periplasmic Hg(2+)-binding protein, MerP, have been examined. While Cys-14 and Cys-17 have previously been shown to be Hg(2+)-binding residues, MerP is readily isolated in an inactive Cys-14-Cys-17 disulfide form. In vivo results demonstrated that these cysteine residues are reduced in the periplasm of Hg(2+)-resistant Escherichia coli. Denaturation and redox equilibrium studies revealed that reduced MerP is thermodynamically favored over the oxidized form. The relative stability of reduced MerP appears to be related to the lowered thiol pK(a) (5.5) of the Cys-17 side chain. Despite its much lower pK(a), the Cys-17 thiol is far less accessible than Cys-14, reacting 45 times more slowly with iodoacetamide at pH 7.5. This is reminiscent of proteins such as thioredoxin and DsbA, which contain a similar C-X-X-C motif, except in those cases the more exposed thiol has the lowered pK(a). In terms of MerP function, electrostatic attraction between Hg(2+) and the buried Cys-17 thiolate may be important for triggering the structural change that MerP has been reported to undergo upon Hg(2+) binding. Control of cysteine residue reactivity in heavy metal binding motifs may generally be important in influencing specific metal-binding properties of proteins containing them.     

Proton stoichiometry in the reduction of the FAD and disulfide of Escherichia coli thioredoxin reductase. Evidence for a base at the active site

The oxidation-reduction midpoint potentials, Em, of the FAD and active site disulfide couples of Escherichia coli thioredoxin reductase have been determined from pH 5.5 to 8.5. The FAD and disulfide couples have similar Em values and thus a linked equilibrium of four microscopic enzyme oxidation-reduction states exists. The binding of phenylmercuric acetate to one enzyme form could be monitored which allowed solving the four microscopic Em values. The Em values at pH 7.0 and 12 degrees C of the four couples of thioredoxin reductase are: (S)2-enzyme-FAD/FADH2 = -0.243 V, (SH)2-enzyme-FAD/FADH2 = -0.260 V, (FAD)-enzyme-(S)2/(SH)2 = -0.254 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.271 V. Thus, at pH 7.0, the FAD and disulfide moieties have a 0.017-V negative interaction and Em values which are different by 0.011 V. The delta Em/delta pH of the FAD couples E2m and E3m are about 0.060 V/pH throughout the pH range studied, showing an approximately 2-proton stoichiometry of reduction of the enzyme FAD. The delta Em/delta pH of the disulfide couples E1m and E4m are about 0.052 V/pH from pH 5.5 to 8.5, showing an apparently nonintegral proton stoichiometry of reduction of 1.8 in this pH range. This proton stoichiometry suggests the presence of a base with an ionization behavior that is linked to the oxidation-reduction state of the disulfide. A novel method is presented for determining the pK values on oxidized and reduced enzyme which agrees with the less accurate classical method. The proton stoichiometry results are consistent with the presence of a thiol-base ion pair in which the pK of the base is elevated from 7.6 in disulfide containing enzyme to greater than 8.5 upon forming an ion pair with a thiol anion of pK 7.0 generated upon reduction of the disulfide. The fluorescence of the FAD in thioredoxin reductase decreases as the pH is lowered with a pK of 7.0, direct evidence for a base near the FAD probably distinct from the base interacting with the dithiol.     

Glutathione-thiyl radical scavenging and transferase properties of human glutaredoxin (thioltransferase). Potential role in redox signal transduction

Glutaredoxin (GRx, thioltransferase) is implicated in cellular redox regulation, and it is known for specific and efficient catalysis of reduction of protein-S-S-glutathione-mixed disulfides (protein-SSG) because of its remarkably low thiol pK(a) ( approximately 3.5) and its ability to stabilize a catalytic S-glutathionyl intermediate (GRx-SSG). These unique properties suggested that GRx might also react with glutathione-thiyl radicals (GS(.)) and stabilize a disulfide anion radical intermediate (GRx-SSG), thereby facilitating the conversion of GS(.) to GSSG or transfer of GS(.) to form protein-SSG. We found that GRx catalyzes GSSG formation in the presence of GS-thiyl radical generating systems (Fe(2+)/ADP/H(2)O(2) + GSH or horseradish peroxidase/H(2)O(2) + GSH). Catalysis is dependent on O(2) and results in concomitant superoxide formation, and it is distinguished from glutathione peroxidase-like activity. With the horseradish peroxidase system and [(35)S]GSH, GRx enhanced the rate of GS-radiolabel incorporation into GAPDH. GRx also enhanced the rate of S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase with GSSG or S-nitrosoglutathione, but these glutathionyl donors were much less efficient. Both actin and protein-tyrosine phosphatase-1B were superior substrates for GRx-facilitated S-glutathionylation with GS-radical. These studies characterize GRx as a versatile catalyst, facilitating GS-radical scavenging and S-glutathionylation of redox signal mediators, consistent with a critical role in cellular regulation.     

Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues

The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin reductase (TrxR), and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys(32)-Gly-Pro-Cys(35) thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys(62)-Cys(69) disulfide as well as dimers and multimers. We have discovered that after incubation with H(2)O(2) only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys(69) and Cys(73) were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys(73), which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154-158). The reversible inhibition of human Trx1 activity by H(2)O(2) and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.     

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.     

Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.     

Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional 1H NMR.

A series of two-dimensional (2D) correlated 1H NMR spectra of reduced and oxidized Escherichia coli thioredoxin have been used to probe the effects of pH in the vicinity of the active site, -Cys32-Gly-Pro-Cys35-, using the complete proton resonance assignments available for thioredoxin. In either oxidation state, the majority of residues of the thioredoxin molecule remain unchanged between pH 5.7 and pH 10, as indicated by the identical chemical shifts of the C alpha H, C beta H, and other protons. In reduced thioredoxin, a fairly widespread region around the active-site dithiol is affected by the titration of a group or groups with pKa approximately 7.1-7.4 in 2H2O. Another titration, with pKa approximately 8.4, affects a smaller region of the protein. Oxidized thioredoxin contains a disulfide and no free thiol groups; nevertheless, the proton resonances of many groups in the active-site region were observed to titrate with a pKa of 7.5, probably as a result of an abnormally high pKa value for the carboxyl group of the buried Asp-26 residue. For reduced thioredoxin, the results indicate that Asp-26 is titrating in this pH range, as well as both thiol groups. The new results are strongly suggestive that the mechanism of thioredoxin-catalyzed protein disulfide reduction may be critically dependent on proton transfer as well as electron transfer within the active site.