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Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

CblC is a chaperone that catalyzes removal of the β-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the β-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.

In humans, cobalamin or vitamin B(12) is delivered to two target enzymes via a complex intracellular trafficking pathway comprising transporters and chaperones. CblC (or MMACHC) is a processing chaperone that catalyzes an early step in this trafficking pathway. CblC removes the upper axial ligand of cobalamin derivatives, forming an intermediate in the pathway that is subsequently converted to the active cofactor derivatives. Mutations in the cblC gene lead to methylmalonic aciduria and homocystinuria. Here, we report that nitrosylcobalamin (NOCbl), which was developed as an antiproliferative reagent, and is purported to cause cell death by virtue of releasing nitric oxide, is highly unstable in air and is rapidly oxidized to nitrocobalamin (NO(2)Cbl). We demonstrate that CblC catalyzes the GSH-dependent denitration of NO(2)Cbl forming 5-coordinate cob(II)alamin, which had one of two fates. It could be oxidized to aquo-cob(III)alamin or enter a futile thiol oxidase cycle forming GSH disulfide. Arg-161 in the active site of CblC suppressed the NO(2)Cbl-dependent thiol oxidase activity, whereas the disease-associated R161G variant stabilized cob(II)alamin and promoted futile cycling. We also report that CblC exhibits nitrite reductase activity, converting cob(I)alamin and nitrite to NOCbl. Finally, the denitration activity of CblC supported cell proliferation in the presence of NO(2)Cbl, which can serve as a cobalamin source. The newly described nitrite reductase and denitration activities of CblC extend its catalytic versatility, adding to its known decyanation and dealkylation activities. In summary, upon exposure to air, NOCbl is rapidly converted to NO(2)Cbl, which is a substrate for the B(12) trafficking enzyme CblC.

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

Disturbance of glutathione (GSH) metabolism is a hallmark of numerous diseases, yet GSH functions are poorly understood. One key to this question is to consider its functional compartmentation. GSH is present in the endoplasmic reticulum (ER), where it competes with substrates for oxidation by the oxidative folding machinery, composed in eukaryotes of the thiol oxidase Ero1 and proteins from the disulfide isomerase family (protein disulfide isomerase). Yet, whether GSH is required for proper ER oxidative protein folding is a highly debated question. Recent Advances: Oxidative protein folding has been thoroughly dissected over the past decades, and its actors and their mode of action elucidated. Genetically encoded GSH probes have recently provided an access to subcellular redox metabolism, including the ER.Of the few often-contradictory models of the role of GSH in the ER, the most popular suggest it serves as reducing power. Yet, as a reductant, GSH also activates Ero1, which questions how GSH can nevertheless support protein reduction. Hence, whether GSH operates in the ER as a reductant, an oxidant, or just as a "blank" compound mirroring ER/periplasm redox activity is a highly debated question, which is further stimulated by the puzzling occurrence of GSH in the Escherichia coli periplasmic "secretory" compartment, aside from the Dsb thiol-reducing and oxidase pathways.Addressing the mechanisms controlling GSH traffic in and out of the ER/periplasm and its recycling will help address GSH function in secretion. In addition, as thioredoxin reductase was recently implicated in ER oxidative protein folding, the relative contribution of each of these two reducing pathways should now be addressed. Antioxid. Redox Signal. 27, 1178-1199.

Most pharmacological studies concerning the beneficial effects of organoselenium compounds have focused on their ability to mimic glutathione peroxidase (GPx). However, mechanisms other than GPx-like activity might be involved on their biological effects. This study was aimed to investigate and compare the protective effects of two well known [(PhSe)2 and PhSeZnCl] and two newly developed (MRK Picolyl and MRK Ester) organoselenium compounds against oxidative challenge in cultured neuronal HT22 cells. The thiol peroxidase and oxidase activities were performed using the glutathione reductase (GR)-coupled assay. In order to evaluate protective effects of the organoselenium compounds against oxidative challenge in neuronal HT22 cells, experiments based on glutamate-induced oxytosis and SIN-1-mediated peroxynitrite generation were performed. The thiol peroxidase activities of the studied organoselenium compounds were smaller than bovine erythrocytes GPx enzyme. Besides, (PhSe)2 and PhSeZnCl showed higher thiol peroxidase and lower thiol oxidase activities compared to the new compounds. MRK Picolyl and MRK Ester, which showed lower thiol peroxidase activity, showed higher thiol oxidase activity. Both pre- or co-treatment with (PhSe)2, PhSeZnCl, MRK Picolyl and MRK Ester protected HT22 cells against glutamate-induced cytotoxicity. (PhSe)2 and MRK Picolyl significantly prevented peroxinitrite-induced dihydrorhodamine oxidation, but this effect was observed only when HT22 were pre-treated with these compounds. The treatment with (PhSe)2 increased the protein expression of antioxidant defences (Prx3, CAT and GCLC) in HT22 cells. Taking together, our results suggest that the biological effects elicited by these compounds are not directly related to their GPx-mimetic and thiol oxidase activities, but might be linked to the up-regulation of endogenous antioxidant defences trough their thiol-modifier effects., The funding was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant Nos. 401065/204-6 and 424809/2018-4) and FAPDF (Grant No. 712/2019). L.G.S. was recipient of a PhD fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Holomycin is a member of the dithiolopyrrolone class of secondary metabolites. It contains a cromophore formed by two heterocycles, one of them carrying a disulfide bridge. The holomycin chromophore is also present in thioluthin, aureothricin, the xenorhabdins and the complex thiomarinols. Streptomyces clavuligerus ATCC 27064 contains the holomycin gene cluster (hlm). This antibiotic is formed by a cysteine-activating non-ribosomal peptide synthetase followed by four reduction steps by a set of four different flavoproteins; the intermediate is cyclized by a thiol oxidase and modified by acylation. Holomycin is a broad spectrum antibiotic, reported as antitumoral, acting in vivo on RNA synthesis. It is modified intracellularly by the producer strains by methylation and formation of heterodimers as a way of self-protection. Holomycin might be a lead molecule for the production of new hybrid compounds with higher activity and lower toxicity.

Ascorbate was linked to protein folding a long time ago. At the first level of this connection, it had been shown that ascorbate functions as an essential cofactor in the hydroxylation enzymes involved in collagen synthesis. Although the hydroxylation reactions catalyzed by the members of the prolyl 4-hydroxylase family are considered to be ascorbate dependent, the hydroxylation of proline alone does not need ascorbate. Prolyl 4-hydroxylases participate in two catalytic reactions: one in which proline residues are hydroxylated, while 2-oxoglutarate is decarboxylated and molecular oxygen is consumed. This reaction is ascorbate independent. However, in another reaction, prolyl 4-hydroxylases catalyze the decarboxylation of 2-oxoglutarate uncoupled from proline hydroxylation but still needing molecular oxygen. At this time, ferrous iron is oxidized and the protein is rendered catalytically inactive until reduced by ascorbate. At the second level of the connection, the oxidation and the oxidized form of ascorbate, dehydroascorbate, is involved in the formation of disulfide bonds of secretory proteins. The significance of the dehydroascorbate reductase activity of protein disulfide isomerase was debated because protein disulfide isomerase as a dehydroascorbate reductase was found to be too slow to be the major route for the reduction of dehydroascorbate (and formation of disulfides) in the endoplasmic reticulum lumen. However, very recently, low tissue ascorbate levels and a noncanonical scurvy were observed in endoplasmic reticulum thiol oxidase- and peroxiredoxin 4-compromised mice. This novel observation implies that ascorbate may be involved in oxidative protein folding and creates a link between the disulfide bond formation (oxidative protein folding) and hydroxylation.

Bovine lens aldose reductase is susceptible to a copper-mediated oxidation, leading to the generation of a disulfide bridge with the concomitant incorporation of two equivalents of the metal and inactivation of the enzyme. The metal complexed by the protein remains redox active, being able to catalyse the oxidation of different physiological thiol compounds. The thiol oxidase activity displayed by the enzymatic form carrying one equivalent of copper ion (Cu1-AR) has been characterized. The efficacy of Cu1-AR in catalysing thiol oxidation is essentially comparable to the free copper in terms of both thiol concentration and pH effect. On the contrary, the two catalysts are differently affected by temperature. The specificity of the AR-bound copper towards thiols is highlighted with Cu1-AR being completely ineffective in promoting the oxidation of both low-density lipoprotein and ascorbic acid.

Two related ER oxidation 1 (ERO1) proteins, ERO1α and ERO1β, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1α and ERO1β. The peak amplitude of calcium transients in homozygous Ero1α mutant adult cardiomyocytes was reduced to 42.0 ± 2.2% (n=10, P≤0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of β adrenergic blockade (P≤0.01). In addition, mice lacking ERO1α were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31±0.02 in the mutant (n=20) vs. 0.23±0.03 in the wild type (n=18); P≤0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.—Chin, K. T., Kang, G., Qu, J., Gardner, L. B., Coetzee, W. A., Zito, E., Fishman, G. I., Ron, R. The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load.

Design and synthesis of organoselenium compounds with high thiol peroxidase (TPx) and low thiol oxidase (TOx) activities have been a difficult task and remains a synthetic-activity relationship dilemma. In this regard we are reporting for the first time a detail experimental data (both in vitro and in vivo) about the anti-oxidant and toxicological profile of an lmine (-N=) containing organoselenium compound (Compound A). The TPx activity of Compound A was significantly higher than diphenyl diselenide (DPDS). Both Compound A and DPDS protected sodium nitropruside (SNP) induced thiobarbituric acid reactive species (TBARS) production in rats tissue homogenate with significantly higher activity observed for Compound A than DPDS (p < 0.05). The Compound A also exhibited strong antioxidant activity in the DPPH and ABTS radical scavenging assays. This study reveals that an imine group close to selenium atom drastically enhances the catalytic activities in the aromatic thiol (PhSH) assay systems. The oxidation of biologically significant thiols reflects the toxicity of the compounds. However, the present data showed that treatment with Compound A at 0, 10.25 or 50 mg/kg was not associated with mortality or body weight loss. Similarly it did not inhibit alpha-ALA-D and Nat(+1)/K+1 ATPase (sulfhydryl group containing enzymes) activities after acute oral treatment; rather it enhanced non-protein thiols (NPSH) concentration. The Compound A did not cause any oxidative stress as measured by TBARS production in rat's tissue preparation. Our data also indicate that exposure to Compound A did not affect plasma transaminase activities or levels of urea and creatinine in rats. Ascorbic acid is always considered a marker of oxidative stress and the reduction of its content may indicate an increase in oxidative stress. Treatment with Compound A did not alter Ascorbic acid levels in rats. The conducted in vitro and in vivo tests show the versatile therapeutic potential of this compound in the area of free radical induced damages, will undoubtedly enhance our understanding of the mechanism of model compounds and may ultimately yield insights that result in improved GPx mimics. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. 1D NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and Cu(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H(2)O(2)) or cysteine in the absence of bicarbonate anion (HCO(3)(-)), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(II) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO(3)(-) protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 Å away from the Cu(II) site, were completely abolished during both HCO(3)(-)-stimulated peroxidase activity and thiol oxidase activity in the presence of HCO(3)(-) . Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO(3)(-) revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO(3)(-), these modifications were absent. HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities.

An enzyme capable of oxidizing thiol groups and tentatively called thiol oxidase has been isolated from barley and malt. There is also considerable incidence of thiol-removing activity in profoundl...

Oxidative folding drives the import of proteins containing twin CXnC motifs into the intermembrane space of mitochondria. This import pathway employs a disulfide relay system whose key components are the redox-regulated import receptor Mia40 and the thiol oxidase Erv1. Mia40 contains six cysteine residues in a CPC-CX9C-CX9C arrangement in a highly conserved domain. We show that this domain is sufficient for the function of Mia40. By analysis of Mia40 cysteine mutants we demonstrate that the cysteine residues have distinct roles and are not equally important for Mia40 function. The second cysteine residue is essential for viability of yeast cells. It is required for the interaction of Mia40 with Erv1 in a disulfide intermediate and forms a redox-sensitive disulfide bond with the first cysteine residue. Both cysteine residues are required for the oxidation of the substrate, Tim10, in a reconstituted system comprised of Mia40 and Erv1. Mutants with amino acid exchanges in the third and sixth cysteine residues have severe defects in growth and in the import of intermembrane space proteins. These Mia40 variants are not tightly folded. We conclude that the cysteine residues of the twin CX9C motif have a structural role and stabilize Mia40. In particular, the disulfide bond formed by the third and sixth cysteine residues apparently supports a conformation crucial for the function of Mia40. Furthermore, the disulfide bond in the CPC segment mediates the redox reactions with the thiol oxidase Erv1 and substrate proteins in mitochondria.

Background Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes. Results We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. Conclusions This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0658-0) contains supplementary material, which is available to authorized users.

Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.

Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.

Activities of oxygen-dependent thiol: O2 oxidoreductase (EC 1.8.3.2) and glutathione-dependent thiol: protein-disulfide oxidoreductase (EC 1.8.4.2) as well as technological value of seven soft spring wheat cultivars grown on different soil under contrasting climatic conditions (Krasnodar krai and Irkutsk oblast) were studied. It was found that the ratio of these enzymatic activities correlated positively with dough physical properties and flour bread-baking quality.

The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10(-7) m cytoplasmic Ca2+. The thiol specific-lipophilic-4,4'-dithiodipyridine (4,4'-DTDP, 1 mm), as well as the hydrophilic thimerosal (1 mm), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one ( approximately 2 msec) to three (approximately 1 msec; approximately 7 msec; approximately 15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (approximately 75 msec) appeared with cis 4, 4'-DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.

Rhodnius prolixus is a blood feeding triatomine bug that contains salivary nitric oxide bound to hemoproteins previously named nitrophorins. Nitrophorins, in addition to storing and transporting NO, have two other functions such as anti-histaminic and anti-clotting (displayed by nitrophorin 2 only). Additionally, nitrophorins display a thiol oxidase reaction, where cysteine is oxidized to cystine with the production of hydrogen peroxide. In this paper the heme-peroxidase reaction of nitrophorins is described. The heme moiety of nitrophorins is destroyed by addition of cysteine or hydrogen peroxide. No biliverdin is produced during this reaction. We have also found that during the thiol oxidase reaction, nitrophorins can destroy norepinephrine, conferring an additional vasodilatory competence for this class of salivary molecules.

Mia40 catalyses the oxidative folding of disulphide-containing proteins in the mitochondria. The folding pathway is directed by the formation of the first mixed disulphide between Mia40 and its substrate. Here, we employ Cox17 to elucidate the molecular determinants of this reaction. Mia40 engages initially in a dynamic non-covalent enzyme–substrate complex that forms and dissociates within milliseconds. Cys36 of Cox17 forms the mixed disulphide in an extremely rapid reaction that is limited by the preceding complex formation with Mia40. Cys36 reacts much faster than the three other cysteines of Cox17, because it neighbours three hydrophobic residues. Mia40 binds preferentially to hydrophobic regions and the dynamic nature of the non-covalent complex allows rapid reorientation for an optimal positioning of the reactive cysteine. Mia40 thus uses the unique proximity between its substrate-binding site and the catalytic disulphide to select a particular cysteine for forming the critical initial mixed disulphide. Proteins containing multiple disulphide bonds fold more efficiently if these bonds are formed in a defined order. Koch et al.find that the mitochondrial thiol oxidase Mia40 achieves this by preferentially targeting cysteine residues within a hydrophobic context.

An NADH oxidase activity of the plasma membrane that was growth factor- and hormone-stimulated has been reported and characterized extensively. The activity is constitutively activated in cancer. Thiol groups are important to its activity and its activity as a thiol oxidoreductase or thiol interchange activity may be important to its functioning in growth and membrane trafficking. The activity is inhibited in the presence of GDP and brefeldin A and appears to be found not only at the plasma membrane but also with the Golgi apparatus. The phenotype of an inhibited NADH oxidase is a failure of cells to enlarge. The hypothesis under investigation is that the hormone- and growth factor-stimulated NADH (quinone)/thiol oxidase functions as an essential component of physical membrane displacements associated with cell elongation, vesicle budding and pleomorphic membrane changes in both animal and plant cells.

Bombyx mori nucleopolyhedrovirus (BmNPV) triggers the global shutdown of host silkworm gene expression and protein synthesis approximately 12–18 h post-infection. Genome sequence analysis suggests that BmNPV ORF75 could be a flavin adenine dinucleotide (FAD)-linked thiol oxidase essential for virion assembly and virus propagation. Here, we report the crystal structure of BmNPV ORF75 at 2.1 Å (0.21 nm). The structure of BmNPV ORF75 resembles that of the thiol oxidase domain of human quiescin thiol oxidase (QSOX), displaying a pseudo-dimer of canonical and non-canonical thiol oxidase domains. However, BmNPV ORF75 is further dimerized by its C-terminal canonical thiol oxidase domain. Within the unique quaternary structural arrangement, the FAD-binding pocket and the characteristic CXXC motif from each monomer is 35 Å (3.5 nm) away from that of its corresponding molecule, which suggests that BmNPV ORF75 might adopt a deviant mechanism from that of QSOX to catalyse disulfide bond formation. Our thiol oxidase activity assay on the point mutations of the conserved residues participating in FAD recognition reveals an aromatic cage next to the FAD isoalloxazine moiety for substrate binding. These data suggest that the thiol oxidase activity of BmNPV ORF75 could be critical to catalyse the formation of the disulfide bonds of certain BmNPV proteins essential for BmNPV virion assembly.

Summary Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy., Graphical Abstract Highlights ► Abnormal extracellular matrix in mice compromised in their ER thiol oxidases ► Abnormal procollagen maturation responsive to ascorbate repletion ► Tissue ascorbate depletion and noncanonical scurvy in the mutant mice

The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in ALR.

The formation of disulfide bonds in proteins and small molecules can greatly affect their functionality. Sulfhydryl oxidases (SOXs) are enzymes capable of oxidising the free sulfhydryl groups in proteins and thiol-containing small molecules by using molecular oxygen as an electron acceptor. SOXs have been isolated from the intracellular compartments of many organisms, but also secreted SOXs are known. These latter enzymes are generally active on small compounds and their physiological role is unknown, whereas the intracellular enzymes prefer proteins as substrates and are involved in protein folding. An increasing number of scientific publications and patent applications on SOXs have been published in recent years. The present mini-review provides an up-to-date summary of SOXs from various families, their production and their actual or suggested applications. The sequence features and domain organisation of the characterised SOXs are reviewed, and special attention is paid to the physicochemical features of the enzymes. A review of patents and patent applications regarding this class of enzymes is also provided.

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 microM) < or = B (0.40 microM) < D (0.68 microM) < A (2.90 microM), for Fe(2+)/EDTA, and B (0.21 microM) < or = C (0.33 microM) < or = D (0.43 microM) < A (1.21 microM) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

A biotinylation-bioselective adsorption procedure was developed for single-step purification of bovine sulfhydryl oxidase from solubilized skim milk membrane vesicles. Sulfhydryl oxidase was specifically biotinylated by reaction of its chemically reactive sulfhydryl group with a disulfide-containing biotinylation reagent giving a mixed disulfide between the enzyme and the biotinyl moiety. The biotinylated enzyme was then selectively adsorbed on a monomeric avidin matrix. After washing all nonspecifically bound proteins from the matrix, the enzyme was released by reduction of the disulfide with dithiothreitol. Both the biotinylation and the isolation are performed at pH 7.0 under extremely mild conditions. The resulting enzyme preparation appeared to be homogeneous by gel electrophoresis and the specific activity increased over 3000 fold in comparison to whey. The monomeric avidin matrix can be regenerated and used indefinitely. This purification procedure should be generally applicable to proteins with a chemically reactive sulfhydryl group.

The oxidation of cysteine by plant peroxidase has been studied. Hydrogen peroxide was identified as a reaction product and measured quantitatively. The amount of hydrogen peroxide generated depends on the concentration of cysteine as well as on the concentration of the enzyme. The maximum rate of hydrogen peroxide production was observed at a cysteine concentration of ca 500 μM depending on the enzyme concentration. At concentrations above this value, cysteine acts more as an antioxidant, while below it acts as a pro-oxidant. With low enzyme concentration the reaction is biphasic and influenced by superoxide dismutase. Horseradish peroxidase was used in most of the experiments, but wood peroxidases from several trees were also studied. In all cases the peroxidases function as thiol oxidases. A reaction mechanism is proposed and the possible physiological role of the reaction, mainly in respect to hydrogen peroxide biosynthesis in plants, is discussed.

(S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate, a new telluroamino acid derivative, showed remarkable glutathione peroxidase (GPx)-like activity, attesting to its antioxidant potential. However, the stability and toxicity of this compound has not yet been investigated. The present study was designed to investigate the pharmacological/toxicological properties of this compound in vitro and in vivo. In vitro, this telluroamino acid derivative significantly blocked spontaneous and Fe(II)-induced TBARS formation in rat brain homogenates, demonstrating high antioxidant activity. In addition, it exhibited GPx-like and thiol oxidase activities. However, when subcutaneously administered to mice, (S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate indicated genotoxic and mutagenic effect in adult male mice. Considering the differential effects of (S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate in vitro and in vivo, additional experiments are needed to elucidate the mechanism(s) by which this compound displays its antioxidant/toxicological effects.

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.

The study, addressed to understand whether or not human platelets possess a unique thiol-oxidase whose activity could be modulated by signalling pathway initiated upon the activation of Receptor-‘C’k revealed the existence of disulphide-dependent oxidation within these cells and this phenomenon was regulated by Receptor-‘C’k-dependent generation of second messengers especially phosphatidic acid (PA); cAMP and cGMP. Purification of this activity revealed the existence of 47 kDa protein having thiol-oxidase activity. Keeping in view these results we propose that the existence of this novel 47 kDa Thiol-oxidase within human platelets may provide a ‘crucial switch’ for the regulation of Receptor-‘C’k-dependent mevalonate pathway in human platelets.

Sulfhydryl oxidase (SOx), an enzyme that catalyzes the oxidation of sulfhydryl compounds, appears in the spermatogenic cells of rat and hamster testes in a stage-dependent manner. It first appears in pachytene spermatocytes at stage I in both the animal species studied. SOx immunoreactivity is associated with mitochondria of these cells. The fate of such mitochondria is species-dependent. In rat, the immunoreactive mitochondria aggregate during maturation phase and are retained in the residual bodies. Spermatozoa free of SOx are released into the lumen. On the other hand, in hamster, the immunoreactive mitochondria arrange themselves around the midpiece of spermatozoa. In such a case, residual bodies lack SOx. The appearance of SOx coincides with the appearance of LDH-X in the spermatogenic cells. Like many other proteins such as LDH-X, RSA-1 and cytochrome ct, SOx provides yet another example of differential gene activation associated with a developmental process of gametes.

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX3C” or twin “CX9C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX2C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.

A thiol oxidase was purified from porcine kidney cortex by chromatography of detergent-solubilized plasma membranes on cysteinylsuccinamidopropyl-glass beads, hydroxyapatite, and Sephacryl S-200. The oxidase was purified 2600-fold; 28% recovery of activity was obtained. With glutathione as substrate, the apparent Km was 0.73 mM and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O2----RSSR + H2O2, and superoxide production was not detected during the reaction. Other low molecular weight thiols, including cysteine, dithiothreitol, N-acetylcysteine, and cysteamine, were substrates for the oxidase; 2-mercaptoethanol, reductively denatured ribonuclease A, and chymotrypsinogen A were not substrates. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band corresponding to 70 kDa; gel filtration on a Sephacryl column produced a single elution of activity with a protein corresponding to 120 kDa, indicating that the functional form is a dimer. On a high-pressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at greater than 200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), H2O2, and N-ethylmaleimide, suggesting the presence of copper and a sulfhydryl group at the active site. Following treatment with metal chelators, enzyme activity was reconstituted with CuSO4, but not with FeSO4. The purified enzyme contained 1 mol copper per subunit which was undetectable by electron paramagnetic resonance, suggesting that the copper is in a binuclear complex.

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Morever, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N -acetylcystein could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/ N -acetylecysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni uni ping-pong mechanism proposed previously for sulfhydryl oxidase.

A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The membrane-associated thiol oxidase of rat small-intestinal epithelium was studied to determine its subcellular localization and properties. The brush-border and basallateral regions of the plasma membrane were isolated by density-gradient centrifugation in Percoll. The intestinal oxidase was localized by use of marker enzymes to the basallateral region of the plasma membrane. The reaction stoichiometry and activity with a variety of low-molecular-weight thiols were determined. The oxidase activity was inhibited by EDTA, bathocuproine disulfonate, N -ethylmaleimide, and H 2 O 2 ; this suggests that copper and a sulfhydryl group are involved in catalysis. Oxidase activity in EDTA-treated basal-lateral membranes was reconstituted with CuSO 4 , which suggests the requirement for copper. These results show that the intestinal oxidase is very similar to the renal oxidase, and because of the subcellular localization and accessibility to extracellular thiols, suggests that the intestinal oxidase may be important in the maintenance of the plasma thiol:disulfide ratio.

Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp95, no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. Our results showed that Erv1W95F has the strongest effect on the stability and function of Erv1 and followed by Erv1W183F. Erv1W95F results in a decrease in the Tm of Erv1 by 23°C, a significant loss of the oxidase activity and thus causing cell growth defects at both 30°C and 37°C. Erv1W183F induces changes in the oligomerization state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whereas the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp157 mutant. Finally, computational analysis indicates that Trp95 plays a key role in stabilizing the isoalloxazine ring to interact with Cys133. Taken together, the present study provided important insights into the molecular mechanism of how thiol oxidases use FAD in catalysing disulfide bond formation.