Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.     

A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase

A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins

Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family. Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs. E. coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E. coli Prx members. Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution. The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues. The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact. Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs. EcTpx exists as a dimer stabilized by hydrogen bonds. Its substrate binding site extends to the dimer interface. A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity.     

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.     

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.     

The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species‐scavenging enzyme that shows promiscuity in thiol redox control

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (‐SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H₂O₂ reduction, a sulfenic acid (‐SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S‐S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx‐MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H₂O₂ stress, and its gene expression is clearly induced upon H₂O₂ challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.     

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.     

Disulfide between Cys392 and Cys438 of human serum albumin is redox-active, which is responsible for the thioredoxin-supported lipid peroxidase activity

Human serum albumin (HSA) is an abundant protein found in blood plasma and extracellular fluids. Previously, we found that HSA has a distinct thioredoxin (Trx)-dependent lipid peroxidase activity in the presence of palmitoyl-CoA. In this paper, we identified the redox-active disulfide, which can be specifically reduced by Trx, responsible for the Trx-dependent lipid peroxidase activity. The IIB-III fragment of HSA (Pro299-Leu585) sustained the Trx-dependent lipid peroxidase activity. Chemical modification of the Trx-reduced IIB-III with a thiol-specific modification agent resulted in a complete loss of the peroxidase activity. The analysis of tryptic-peptides derived from the inactivated HSA and IIB-III revealed that Cys392 and Cys438, which exist as an intramolecular disulfide bond in HSA, were preferentially modified in both HSA and IIB-III. Taken together, these results suggested that HSA has a capability to reduce lipid hydroperoxide with the use of Trx as an in vivo electron donor, and that the redox-active disulfide between Cys392 and Cys438 acts as a primary site of the catalysis for the Trx-linked lipid peroxidase activity.     

Kinetic and thermodynamic features reveal that Escherichia coli BCP is an unusually versatile peroxiredoxin

In Escherichia coli, bacterioferritin comigratory protein (BCP) is a peroxiredoxin (Prx) that catalyzes the reduction of H(2)O(2) and organic hydroperoxides. This protein, along with plant PrxQ, is a founding member of one of the least studied subfamilies of Prxs. Recent structural data have suggested that proteins in the BCP/PrxQ group can exist as monomers or dimers; we report here that, by analytical ultracentrifugation, both oxidized and reduced E. coli BCP behave as monomers in solution at concentrations as high as 200 μM. Unexpectedly, thioredoxin (Trx1)-dependent peroxidase assays conducted by stopped-flow spectroscopy demonstrated that V(max,app) increases with increasing Trx1 concentrations, indicating a nonsaturable interaction (K(m) > 100 μM). At a physiologically reasonable Trx1 concentration of 10 μM, the apparent K(m) value for H(2)O(2) is ~80 μM, and overall, the V(max)/K(m) for H(2)O(2), which remains constant at the various Trx1 concentrations (consistent with a ping-pong mechanism), is ~1.3 × 10(4) M(-1) s(-1). Our kinetic analyses demonstrated that BCP can utilize a variety of reducing substrates, including Trx1, Trx2, Grx1, and Grx3. BCP exhibited a high redox potential of -145.9 ± 3.2 mV, the highest to date observed for a Prx. Moreover, BCP exhibited a broad peroxide specificity, with comparable rates for H(2)O(2) and cumene hydroperoxide. We determined a pK(a) of ~5.8 for the peroxidatic cysteine (Cys45) using both spectroscopic and activity titration data. These findings support an important role for BCP in interacting with multiple substrates and remaining active under highly oxidizing cellular conditions, potentially serving as a defense enzyme of last resort.     

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.     

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.     

How Thioredoxin Dissociates Its Mixed Disulfide

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29^Trx-Cys89^ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29^Trx on the exposed Cys82^ArsC-Cys89^ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32^Trx in contact with Cys29^Trx. Cys32^Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32^Trx is found to be more reactive than Cys82^ArsC. Additionally, Cys32^Trx directs its nucleophilic attack on the more susceptible Cys29^Trx and not on Cys89^ArsC. This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.     

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.     

An atypical catalytic mechanism involving three cysteines of thioredoxin

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys(4)) and two (Cys(58) and Cys(61)) in the classical thioredoxin active site ((57)WCGPC(61)). The property of a mutant in which Cys(58) was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys(4) is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys(58) can be formed, linking it to either Cys(61) or Cys(4). We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys(4) to convert this atypical thioredoxin h back to its active reduced form.     

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.