Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.     

Atomic-resolution crystal structure of thioredoxin from the acidophilic bacterium Acetobacter aceti

The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 A resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization.     

Regulation of protein function by glutathionylation

The main function of reduced glutathione (GSH) is to protect from oxidative stress as a reactive oxygen scavenger. However, in the context of redox regulation, the ratio between GSH and its oxidized form (GSSG) determines the redox state of redox-sensitive cysteines in some proteins and, thus, acts as a signaling system. While GSH/GSSG can catalyze oxido-reduction of intra- and inter-chain disulfides by thiol-disulfide exchange, this review focuses on the formation of mixed disulfides between glutathione and proteins, also known as glutathionylation. The review discusses the regulatory role of this post-translational modification and the role of protein disulfide oxidoreductases (thioredoxin/thioredoxin reductase, glutaredoxin, protein disulfide isomerase) in the reversibility of this process.     

Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats

Rabbit antisera against homogeneous rat liver thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) were prepared and used for immunohistochemical analysis in adult rats. Immunoreactive thioredoxin and thioredoxin reductase were widely distributed in tissues and organs, but varied a lot between cell types. Generally, epithelial cells, neuronal cells and secretory cells, both exocrine and endocrine, showed high immunoreactivity whereas mesenchymal cells with exceptions showed low activity. Surface lining epithelial and keratinizing cells showed high activity. The immunofluorescence was localized in the cytoplasm of cells with enrichments at secretory granules, at the plasma membrane or in the subplasma membrane zone. Variations in secretory cells were seen related to feeding and starvation and to metabolic activity. The distribution of thioredoxin and thioredoxin reductase is compatible with function in thiol-disulfide interchange reaction related to protein synthesis, intracellular transport and different forms of secretion.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

Crystal structure of DsbDgamma reveals the mechanism of redox potential shift and substrate specificity(1)

The Escherichia coli transmembrane protein DsbD transfers electrons from the cytoplasm to the periplasm through a cascade of thiol-disulfide exchange reactions. In this process, the C-terminal periplasmic domain of DsbD (DsbDgamma) shuttles the reducing potential from the membrane domain (DsbDbeta) to the N-terminal periplasmic domain (DsbDalpha). The crystal structure of DsbDgamma determined at 1.9 A resolution reveals that the domain has a thioredoxin fold with an extended N-terminal stretch. In comparison to thioredoxin, the DsbDgamma structure exhibits the stabilized active site conformation and the extended active site alpha2 helix that explain the domain's substrate specificity and the redox potential shift, respectively. The hypothetical model of the DsbDgamma:DsbDalpha complex based on the DsbDgamma structure and previous structural studies indicates that the conserved hydrophobic residue in the C-X-X-C motif of DsbDgamma may be important in the specific recognition of DsbDalpha.     

Linked thioredoxin-glutathione systems in platyhelminths

The thioredoxin and glutathione systems play a central role in thiol-disulfide redox homeostasis in many organisms by providing electrons to essential enzymes, and defence against oxidative stress. These systems have recently been characterized in platyhelminth parasites, and the emerging biochemical scenario is the existence of linked processes with the enzyme thioredoxin glutathione reductase supplying reducing equivalents to both pathways. In contrast to their hosts, conventional thioredoxin reductase and glutathione reductase enzymes appear to be absent. Analysis of published data and expressed-sequence tag databases indicates the presence of linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of these parasites.     

A new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120

In silico analysis followed by experimental validation leads us to propose that the predicted protein All0195 of Anabaena sp. PCC7120 showing enhanced expression under sodium arsenate (Na₂HAsO₄) stress belongs to the thioredoxin superfamily with structural similarity to bacterial arsenate reductase. The All0195 protein demonstrated C-X-TC-X-K, NTSG-X₂-YR, and D-X₂-L-X-KRP as functional motifs that show similarity to seven known bacterial arsenate reductase family protein homologs with Cys, Arg, and Pro as conserved residues. In view of physicochemical properties, such as aliphatic index, ratio of Glu?+?Lys to Gln?+?His, and secondary structure, it was evident that All0195 was also a thermostable protein. The predicted three-dimensional structure on molecular docking with arsenate oxyanion ( $ HAsO_4^{- 2 } $ ) revealed its interaction with conserved Cys residue as also known for other bacterial arsenate reductase. In silico derived properties were experimentally attested by cloning and heterologous expression of all0195. Furthermore, this protein functionally complemented the arsenate reductase-deficient sodium arsenate-hypersensitive phenotype of Escherichia coli strainWC3110 (ΔarsC) and depicted arsenate reductase activity on purification. In view of the above properties, All0195 appears to be a new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120.     

Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen.

The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox-active dithiols. We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 microM selenite plus 0.05 microM calf thymus thioredoxin reductase at pH 7.5 caused a non-stoichiometric oxidation of NADPH (100 microM after 30 min). In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 microM E. coli thioredoxin also resulted in non-stoichiometric oxidation of NADPH, consistent with oxidation of the two active-site thiol groups in thioredoxin to a disulfide. Kinetically, the reaction was complex with a lag phase at low selenite concentrations. Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s). Ferricytochrome c was effectively reduced by calf thymus thioredoxin reductase and selenite in the presence of oxygen. Selenite caused a strong dose-dependent inhibition of the formation of thiol groups from insulin disulfides with either the E. coli or calf-thymus thioredoxin system. Thus, under aerobic conditions selenite catalyzed, NADPH-dependent redox cycling with oxygen, a large oxygen-dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide-reductase activity.     

Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.     

The effect of helix-coil transition on backbone 15N NMR relaxation of isolated transmembrane segment (1–36)-bacteriorhodopsin

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b and a. Both a and a domains contain an active site with the sequence motif -Cys-Gly-His-Cys- involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol., 7, 239–245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.     

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.     

Solution structures and backbone dynamics of arsenate reductase from Bacillus subtilis: reversible conformational switch associated with arsenate reduction

Arsenate reductase encoded by the chromosomal arsC gene in Bacillus subtilis catalyzes the intracellular reduction of arsenate to arsenite, which is then extruded from cells through an efficient and specific transport system. Herein, we present the solution structures and backbone dynamics of both the reduced and oxidized forms of arsenate reductase from B. subtilis. The overall structures of both forms are similar to those of bovine low molecular weight protein-tyrosine phosphatase and arsenate reductase from Staphylococcus aureus. However, several features of the tertiary structure and mobility are notably different between the reduced and oxidized forms of B. subtilis arsenate reductase, particularly in the P-loop region and the segment Cys(82)-Cys(89). The backbone dynamics results demonstrated that the reduced form of arsenate reductase undergoes millisecond conformational changes in the functional P-loop and Cys(82)-Cys(89), which may facilitate the formation of covalent intermediates and subsequent reduction of arsenate. In the oxidized form, Cys(82)-Cys(89) shows motional flexibility on both picosecond-to-nanosecond and possibly millisecond time scales, which may facilitate the reduction of the oxidized enzyme by thioredoxin to regenerate the active enzyme. Overall, the internal dynamics and static structures of the enzyme provide insights into the molecular mechanism of arsenate reduction, especially the reversible conformational switch and changes in internal motions associated with the catalytic reaction.     

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.     

Kinetics of electron transfer from thioredoxin reductase to thioredoxin

The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

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 -165mV 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 -220mV 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? 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.     

Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase.

Selenium compounds like selenite (SeO3(2-) may form a covalent adduct with glutathione (GSH) in the form of selenodiglutathione (GS-Se-SG), which is assumed to be important in the metabolism of selenium. We have isolated GS-Se-SG and studied its reactions with NADPH and thioredoxin reductase from calf thymus or with thioredoxin reductase and thioredoxin from Escherichia coli. Incubation of 0.1 microM calf thymus thioredoxin reductase or 0.1 microM thioredoxin reductase and 1 microM thioredoxin from E. coli with 5, 10, or 20 microM GS-Se-SG resulted in a fast initial reaction, followed by a large and continued oxidation of NADPH. However, anaerobic incubation of 0.1 microM calf thymus thioredoxin reductase and 20 microM GS-Se-SG resulted only in oxidation of a stoichiometric amount of NADPH; admission of oxygen started continuous NADPH oxidation. Contrary to the mammalian enzyme, GS-Se-SG was not a substrate for thioredoxin reductase from E. coli. The rate of the oxygen-dependent reaction between calf thymus thioredoxin reductase and GS-Se-SG was increased 2-fold in the presence of 4 mM GSH, indicating that HSe- was the reactive intermediate. Glutathione reductase from rat liver reduced GS-Se-SG with a very slow continued oxidation of NADPH, and the presence of the enzyme did not affect the oxygen-dependent nonstoichiometric oxidation of NADPH by GS-Se-SG and thioredoxin reductase. Fluorescence spectroscopy showed GS-Se-SG to be a very efficient oxidant of reduced thioredoxin from E. coli and kinetically superior to insulin disulfides. Thioredoxin-dependent reduction of CDP to dCDP by ribonucleotide reductase was effectively inhibited by GS-Se-SG.     

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.     

An arsenate reductase from Synechocystis sp. strain PCC 6803 exhibits a novel combination of catalytic characteristics

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V(max) = 3.1 micro mol/min. mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V(max) = 0.08 micro mol/min. mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.