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With the discovery of glutathione peroxidase (GPx1), the role of glutathione in counteracting oxidative challenge became clear. GPx1 was the first selenoprotein discovered in mammals. It contains a selenocysteine residue integrated into the peptide chain. The phospholipid hydroperoxide glutathione peroxidase (GPx4) also proved to be a selenoprotein. In the cytosol, it inhibits lipid peroxidation and ferroptosis; in the nucleus, it supports protamine compaction; its mitochondrial expression form builds the sheath surrounding the mitochondria in spermatozoa and is essential for male fertility. In the meantime, glutathione peroxidases have grown into a large family of enzymes that work with selenium or with sulfur catalysis. With the growing knowledge that hydroperoxides are not just toxic, but normal intermediates of biosynthetic processes and signaling molecules, glutathione peroxidases had to be re-considered as regulatory proteins in signaling cascades, as modulators of enzyme activities, or as hydrogen peroxide sensors. The extraordinary catalytic efficiency of both the selenium- and sulfur-containing enzymes remained enigmatic for a long time. Recent quantum mechanical approaches revealed a concerted dual attack on the peroxide bond, which explains why their speed of peroxide reduction is orders of magnitude higher than that of any low molecular weight thiol or selenol.

The discoveries leading to our present understanding of the glutathione peroxidases (GPxs) are recalled. The cytosolic GPx, now GPx1, was first described by Mills in 1957 and claimed to depend on selenium by Rotruck et al., in 1972. With the determination of a stoichiometry of one selenium per subunit, GPx1 was established as the first selenoenzyme of vertebrates. In the meantime, the GPxs have grown up to a huge family of enzymes that prevent free radical formation from hydroperoxides and, thus, are antioxidant enzymes, but they are also involved in regulatory processes or synthetic functions. The kinetic mechanism of the selenium-containing GPxs is unusual in neither showing a defined K

The so-called peroxidatic cysteines and selenocysteines in proteins reduce hydroperoxides through a dual attack to the peroxide bond in a two-step mechanism. First, a proton dislocation from the thiol/selenol to a close residue of the enzymatic pocket occurs. Then, a nucleophilic attack of the anionic cysteine/selenocysteine to one O atom takes place, while the proton is shuttled back to the second O atom, promoting the formation of a water molecule. In this computational study, we use a molecular model of GPx to demonstrate that the enzymatic environment significantly lowers the barrier of the latter SN2 step. Particularly, in our Se-based model the energy barriers for the two steps are 29.82 and 2.83 kcal mol−1, both higher than the corresponding barriers computed in the enzymatic cluster, i. e., 21.60 and null, respectively. Our results, obtained at SMD-B3LYP-D3(BJ)/6-311+G(d,p), cc-pVTZ//B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ level of theory, show that the mechanistic details can be well reproduced using an oversimplified model, but the energetics is definitively more favorable in the GPx active site. In addition, we pinpoint the role of the chalcogen in the peroxide reduction process, rooting the advantages of the presence of selenium in its acidic and nucleophilic properties.

The so-called peroxidatic cysteines and selenocysteines in proteins reduce hydroperoxides through a dual attack to the peroxide bond in a two-step mechanism. First, a proton dislocation from the thiol/selenol to a close residue of the enzymatic pocket occurs. Then, a nucleophilic attack of the anionic cysteine/selenocysteine to one O atom takes place, while the proton is shuttled back to the second O atom, promoting the formation of a water molecule. In this computational study, we use a molecular model of GPx to demonstrate that the enzymatic environment significantly lowers the barrier of the latter S

The beginnings of redox biology are recalled with special emphasis on formation, metabolism and function of reactive oxygen and nitrogen species in mammalian systems. The review covers the early history of heme peroxidases and the metabolism of hydrogen peroxide, the discovery of selenium as integral part of glutathione peroxidases, which expanded the scope of the field to other hydroperoxides including lipid hydroperoxide, the discovery of superoxide dismutases and superoxide radicals in biological systems and their role in host defense, tissue damage, metabolic regulation and signaling, the identification of the endothelial-derived relaxing factor as the nitrogen monoxide radical and its physiological and pathological implications. The article highlights the perception of hydrogen peroxide and other hydroperoxides as signaling molecules, which marks the beginning of the flourishing fields of redox regulation and redox signaling. Final comments describe the development of the redox language. In the 18th and 19th century, it was highly individualized and hard to translate into modern terminology. In the 20th century, the redox language co-developed with the chemical terminology and became clearer. More recently, the introduction and inflationary use of poorly defined terms has unfortunately impaired the understanding of redox events in biological systems.

The (seleno)cysteine residues in some protein families react with hydroperoxides with rate constants far beyond those of fully dissociated low molecular weight thiol or selenol compounds. In case of the glutathione peroxidases, we could demonstrate that high rate constants are achieved by a proton transfer from the chalcogenol to a residue of the active site [Orian et al. Free Radic. Biol. Med. 87 (2015)]. We extended this study to three more protein families (OxyR, GAPDH and Prx). According to DFT calculations, a proton transfer from the active site chalcogenol to a residue within the active site is a prerequisite for both, creating a chalcogenolate that attacks one oxygen of the hydroperoxide substrate and combining the delocalized proton with the remaining OH or OR, respectively, to create an ideal leaving group. The “parking postions” of the delocalized proton differ between the protein families. It is the ring nitrogen of tryptophan in GPx, a histidine in GAPDH and OxyR and a threonine in Prx. The basic principle, however, is common to all four families of proteins. We, thus, conclude that the principle outlined in this investigation offers a convincing explanation for how a cysteine residue can become peroxidatic., Graphical abstract Image 1, Highlights • In some protein families, (seleno)cysteine residues react with hydroperoxides with very high rate constants. • In GPx, DFT models of the oxidation of the catalytic site support a two-step mechanism for the H2O2 reduction. • This mechanism is here found to operate in other thiol-based enzymes, i.e. OxyR, GAPDH and Prx.

Significance: The selenium-containing Glutathione peroxidases (GPxs)1-4 protect against oxidative challenge, inhibit inflammation and oxidant-induced regulated cell death. Recent Advances: GPx1 and GPx4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides. GPx2 regulates the balance between regeneration and apoptotic cell shedding in the intestine. It inhibits inflammation-induced carcinogenesis in the gut but promotes growth of established cancers. GPx3 deficiency facilitates platelet aggregation likely via disinhibition of thromboxane biosynthesis. It is also considered a tumor suppressor. GPx4 is expressed in three different forms. The cytosolic form proved to inhibit interleukin-1-driven nuclear factor κB activation and leukotriene biosynthesis. Moreover, it is a key regulator of ferroptosis, because it reduces hydroperoxy groups of complex lipids and silences lipoxygenases. By alternate substrate use, the nuclear form contributes to chromatin compaction. Mitochondrial GPx4 forms the mitochondrial sheath of spermatozoa and, thus, guarantees male fertility. Out of the less characterized GPxs, the cysteine-containing GPx7 and GPx8 are unique in contributing to oxidative protein folding in the endoplasmic reticulum by reacting with protein isomerase as an alternate substrate. A yeast 2-Cysteine glutathione peroxidase equipped with CP and CR was reported to sense H2O2 for inducing an adaptive response. Critical Issues: Most of the findings compiled are derived from tissue culture and/or animal studies only. Their impact on human physiology is sometimes questionable. Future Directions: The expression of individual GPxs and GPx-dependent regulatory phenomena are to be further investigated, in particular in respect to human health.

Selenium compounds that contain selenol functions or can be metabolized to selenols are toxic via superoxide and H2O2 generation, when ingested at dosages beyond requirement. At supra-nutritional dosages various forms of programmed cell death are observed. At physiological intakes, selenium exerts its function as constituent of selenoproteins, which overwhelmingly are oxidoreductases. Out of those, the glutathione peroxidases counteract hydroperoxide-stimulated signaling cascades comprising inflammation triggered by cytokines or lipid mediators, insulin signaling and different forms of programmed cell death. Similar events are exerted by peroxiredoxins, which functionally depend on the selenoproteins of the thioredoxin reductase family. The thiol peroxidases of both families can, however, also act as sensors for hydroperoxides, thereby initiating signaling cascades. Although the interaction of selenoproteins with signaling events has been established by genetic techniques, the in vivo relevance of these findings is still hard to delineate for several reasons: The biosynthesis of individual selenoproteins responds differently to variations of selenium intakes; selenium is preferentially delivered to privileged tissues via inter-organ trafficking and receptor-mediated uptake, and only half of the selenoproteins known by sequence have been functionally characterized. The fragmentary insights do not allow any uncritical use of selenium for optimizing human health.

The early work of Helmut Sies on mammalian hydroperoxide metabolism is reviewed with particular emphasis on the in situ function of catalase and glutathione peroxidase1. Starting out from a catalase-dominated thinking in the middle of the last century, Sies first demonstrated, by whole organ spectroscopy, that H2O2 is generated in rat liver and metabolized by catalase. In a joined effort with the author's group, he then worked out that glutathione peroxidase can kinetically compete with catalase in hydroperoxide metabolism in situ. In compartmentalized cells, however, the "competition" of the two enzymes turned out to be a mutual complementation because of their different subcellular location. The studies for the first time documented that the metabolism of freely diffusible hydroperoxides is compartmentalized and, thus, paved the way to a better understanding of oxidant challenges and redox regulation. The article, garnished with personal memories, is meant as a nostalgic journey though ancient times of biochemistry with their changing fashions and paradigms, revealing the roots of topical perspectives and controversies in redox biology.

The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H2O2 for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10(5)-10(8) M(-1) s(-1), thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.

Background The term GSSG/GSH redox potential is frequently used to explain redox regulation and other biological processes. Scope of review The relevance of the GSSG/GSH redox potential as driving force of biological processes is critically discussed. It is recalled that the concentration ratio of GSSG and GSH reflects little else than a steady state, which overwhelmingly results from fast enzymatic processes utilizing, degrading or regenerating GSH. Major conclusions A biological GSSG/GSH redox potential, as calculated by the Nernst equation, is a deduced electrochemical parameter based on direct measurements of GSH and GSSG that are often complicated by poorly substantiated assumptions. It is considered irrelevant to the steering of any biological process. GSH-utilizing enzymes depend on the concentration of GSH, not on [GSH] 2 , as is predicted by the Nernst equation, and are typically not affected by GSSG. Regulatory processes involving oxidants and GSH are considered to make use of mechanistic principles known for thiol peroxidases which catalyze the oxidation of hydroperoxides by GSH by means of an enzyme substitution mechanism involving only bimolecular reaction steps. General significance The negligibly small rate constants of related spontaneous reactions as compared with enzyme-catalyzed ones underscore the superiority of kinetic parameters over electrochemical or thermodynamic ones for an in-depth understanding of GSH-dependent biological phenomena. At best, the GSSG/GSH potential might be useful as an analytical tool to disclose disturbances in redox metabolism. This article is part of a Special Issue entitled Cellular Functions of Glutathione.

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Background The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. Methodology/Principal Finding A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z’ and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. Conclusions/Significance Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors., Author Summary Parasites from the genus Trypanosoma and Leishmania are etiologic agents for a group of neglected diseases with high morbidity and mortality rates in the developing world. Inasmuch as vaccine development is hampered by the successful mechanisms employed by the pathogens to evade the host immune response, chemotherapy remains as a safe option to fight these diseases. However, new drugs with better pharmacological performance (i.e. safety, efficacy and ease of administration) than those in current use are urgently needed. The thiol-redox metabolism of trypanosomatids offers an excellent opportunity for the development of more selective and efficacious medicines because it depends on a molecule, trypanothione (a bis-glutathionyl derivative of spermidine), unique and indispensable to the pathogens. Here we report the identification of novel inhibitors of trypanothione synthetase from three major trypanosomatid species of medical and veterinary relevance. Although highly conserved in sequence, trypanothione synthetases display significant species-specifity towards compounds, pointing to structural differences as determinants of ligand selectivity. Most of the active compounds presented two-digit μM inhibitory activity and serve as primary scaffolds to develop more potent inhibitors. Among them, N,N'-bis(benzyl)-substituted diamine and paullone derivatives are interesting candidates because of their potent and/or selective anti-trypanosomal and anti-trypanothione synthetase activity.

The catalytic mechanism of glutathione peroxidases and its variations in the subfamilies are reviewed and biological roles of the individual enzymes are compiled. The oxidative part of the catalytic cycle involves a water-mediated charge separation in the reaction center leading to dissociation of the selenocysteine (or cysteine) residue and binding of the delocalized proton in a highly energized position. In this environment, a suitably bound H2O2 is cleaved without any energy barrier in a concerted reaction yielding water and a selenenic (sulfenic) acid. Depending on family subtype and physiological conditions, the unstable oxidized enzymes form intramolecular disulfide or selenenylamide bonds. The reductive part of the cycle involves the reaction of selenenic (sulfenic) acid with a thiol and (selena) disulfide exchange. Trivial in principle, the reduction steps are most variable within the family, which explains its diversified specificities ranging from GSH to thioredoxin, disulfide isomerases, and particular SH groups of other proteins. The versatility in substrate and co-substrate use predestines these proteins for redox regulation, either as competitors for hydroperoxide utilization by other regulatory proteins or as sensor(s)/transducer(s) in hydroperoxide-initiated signaling cascades.

Biosynthesis and the use of trypanothione, a redox metabolite of parasitic trypanosomatids, are reviewed here with special emphasis on the development of trypanocidal drugs. This metabolic system is unique to and essential for the protozoal parasites. Selective inhibition of key elements of trypanothione metabolism, therefore, promises eradication of the parasites without affecting the host. Considering the metabolic importance and drugability of system components, inhibition of the enzymes for regeneration and de novo synthesis of trypanothione is rated as the most promising approach, while related peroxidases and redoxins are disregarded as targets because of limited chances to achieve selective inhibition. The organizational need to exploit the accumulating knowledge of trypanosomatid metabolism for medical practice is briefly addressed.

Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.

Kinetics and molecular mechanisms of GPx-type enzymes are reviewed with emphasis on structural features relevant to efficiency and specificity. In Sec-GPxs the reaction takes place at a single redox centre with selenocysteine as redox-active residue (peroxidatic Sec, U(P)). In contrast, most of the non-vertebrate GPx have the U(P) replaced by a cysteine (peroxidatic Cys, C(P)) and work with a second redox centre that contains a resolving cysteine (C(R)). While the former type of enzymes is more or less specific for GSH, the latter are reduced by "redoxins". The common denominator of the GPx family is the first redox centre comprising the (seleno)cysteine, tryptophan, asparagine and glutamine. In this architectural context the rate of hydroperoxide reduction by U(P) or C(P), respectively, is enhanced by several orders of magnitude compared to that of free selenolate or thiolate. Mammalian GPx-1 dominates H(2)O(2) metabolism, whereas the domain of GPx-4 is the reduction of lipid hydroperoxides with important consequences such as counteracting 12/15-lipoxygenase-induced apoptosis and regulation of inflammatory responses. Beyond, the degenerate GSH specificity of GPx-4 allows selenylation and oxidation to disulfides of protein thiols. Heterodimer formation of yeast GPx with a transcription factor is discussed as paradigm of a redox sensing that might also be valid in vertebrates.

Peroxidase research has been instrumental in defining the principles of chemical catalysis. By now, enzymes termed peroxidases represent a heterogeneous group of distinct enzyme families that operate by different catalytic principles and fulfill diverse biological functions, detoxifying H2O2 being just one of many aspects. H2O2 -dependent synthesis of secondary metabolites is the domain of heme peroxidases and related enzymes operating by transition metal catalysis, that often is mediated by free radical formation. Instead, the coenzyme-free glutathione peroxidases and peroxiredoxins only catalyze two-electron transitions and, thus, can reliably remove hydroperoxides without causing radical-mediated collateral damage. However, their ability to use hydroperoxides for the formation of specific disulfide bonds with and within particular proteins broadens their spectrum of biological activities to differentiation phenomena, redox regulation of metabolic processes, redox sensing, and signalling. The present Forum Editorial tries to guide the reader through the 190 years of equally bewildering and fascinating research on peroxidases up to the topical frontiers of the field that are addressed in this issue.

The role of selenium in male fertility is reviewed with special emphasis on selenoprotein P and phospholipid hydroperoxide glutathione peroxidase (GPx4) in spermiogenesis. Inverse genetics reveal that selenoprotein P is required for selenium supply to the testis. GPx4 is abundantly synthesized in spermatids. As a moonlighting protein it is transformed in the later stages of spermiogenesis from an active selenoperoxidase into a structural protein that becomes a constituent of the mitochondrial sheath of spermatozoa. The transformation is paralleled by loss of glutathione. Mechanistically, the process is an alternate substrate inactivation of GPx4 resulting from reactions of its selenenic form with thiols of GPx4 itself and other proteins. Circumstantial evidence and ongoing experimental genetics indicate that the mitochondrially expressed form of the GPx4 gene is the most relevant one in spermiogenesis, with the nuclear form being dispensable for fertility and the role of cytosolic GPx4 remaining unclear. Clinical data reveal a strong association of low sperm GPx4 with infertility. Thus, impaired GPx4 biosynthesis, due to selenium deficiency or to genetic defects in gpx4 itself or in proteins involved in Se distribution and selenoprotein biosynthesis, causes male infertility, but can also be an epiphenomenon due to any perturbation of testicular function.

In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3–7-fold higher in the parasite form infecting mammals (50–100 μM) than in the form hosted by the tsetse fly (7–34 μM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 μM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2–3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.

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.

Glutathione peroxidases (GPxs) are enzymes working with either selenium or sulfur catalysis. They adopted diverse functions ranging from detoxification of H2O2 to redox signaling and differentiation. The relative stability of the selenoenzymes, however, remained enigmatic in view of the postulated involvement of a highly unstable selenenic acid form during catalysis. Nevertheless, density functional theory calculations obtained with a representative active site model verify the mechanistic concept of GPx catalysis and underscore its efficiency. However, they also allow that the selenenic acid, in the absence of the reducing substrate, reacts with a nitrogen in the active site. MS/MS analysis of oxidized rat GPx4 complies with the predicted structure, an 8-membered ring, in which selenium is bound as selenenylamide to the protein backbone. The intermediate can be re-integrated into the canonical GPx cycle by glutathione, whereas, under denaturing conditions, its selenium moiety undergoes ?-cleavage with formation of a dehydro-alanine residue. The selenenylamide bypass prevents destruction of the redox center due to over-oxidation of the selenium or its elimination and likely allows fine-tuning of GPx activity or alternate substrate reactions for regulatory purposes.

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m(-1) s(-1), respectively, at pH 7.4 and 37 degrees C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 x 10(5) and 7.2 x 10(5) m(-1) s(-1) at pH 7.4 and 37 degrees C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn(3+)-porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo.

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.

A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [13C] and [15N] into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [13C]-glucose is achieved, while the approximate amount of the [15N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [13C/15N]-labelled tryparedoxin of Crithidia fasciculata. Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), the product of gpx-4, is the major selenoprotein in sperm and is considered essential for fertilization because of its multiple roles in spermatogenesis, such as hydroperoxide detoxification, formation of the mitochondrial capsule, and chromatin condensation. Genomic DNA sequences of 3.148 kilobases covering the whole gpx-4 and its flanking regions were amplified from 63 men using the polymerase chain reaction and were analyzed for polymorphisms by direct sequencing. A total of 23 variant sites were detected; 2 were present only in control men (proven fathers; n = 21) and 10 were common to fertile controls and infertile patients (n = 42). A further 11 variant sites were seen in five of the infertile men only. Four of the gpx-4 variants were considered irrelevant to GPx-4-related fertility problems because they occurred homozygously in controls. The majority of the remaining variant sites are also of questionable relevance because they are located in introns or, as third base exchanges, do not affect the protein sequence. However, one of the exon variations leads to an Ala93-Thr exchange that reduces activity in a porcine GPx-4 homologue. Two detected promoter variations were shown by reporter gene constructs to affect transcription in somatic cell lines. These results indicate that gpx-4 polymorphism cannot generally account for the correlation of PHGPx content of sperm and fertility-related parameters, but further examination of this gene as a potential cause of infertility in particular cases is warranted.

In Kinetoplastida 2-Cys peroxiredoxins are the ultimate members of unique enzymatic cascades for detoxification of peroxides, which are dependent on trypanothione, a small thiol specific to these organisms. Here we report on two distinct Leishmania infantum peroxiredoxins, Li cTXNPx and Li mTXNPx, that may be involved in such a pathway. Li cTXNPx, found in the cytoplasm, is a typical 2-Cys peroxiredoxin encoded by LicTXNPx , a member of a multicopy gene family. Li mTXNPx, encoded by a single copy gene, Li m TXNPx , is confined to the mitochondrion and is unusual in possessing an Ile-Pro-Cys motif in the distal redox center, replacing the common peroxiredoxin Val-Cys-Pro sequence, apart from an N-terminal mitochondrial leader sequence. Based on sequence and subcellular localization, the peroxiredoxins of Kinetoplastida can be separated in two distinct subfamilies. As an approach to investigate the function of both peroxiredoxins in the cell, L. infantum promastigotes overexpressing Li cTXNPx and Li mTXNPx were assayed for their resistance to H 2 O 2 and tert -butyl hydroperoxide. The results show evidence that both enzymes are active as peroxidases in vivo and that they have complementary roles in parasite protection against oxidative stress.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 6 60.1 units/mg sperm protein vs. 187.5 6 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 6 45.42 units/mg; P , 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r 5 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity. fertilization, gamete biology, male sexual function, sperm, spermatogenesis

Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H2O2, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k(cat) of 11.2 s(-1) and limiting K(m) values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 microM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173' that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis.

Covering: 1934 to early 2002.The biochemistry of selenium-containing natural products, including selenoproteins, is reviewed up to May 2002. Particular emphasis is placed on the assimilation of selenium from inorganic and organic selenium sources for selenoprotein synthesis, the catalytic role of selenium in enzymes, and medical implications of an unbalanced selenium supply. The review contains 393 references on key discoveries and recent progress.

The GSH/GSSG redox potential has become a fashionable electrochemical parameter believed to be a major driving force of redox reactions regulating biological events (Schafer and Buettner, 2001; Jones, 2006; Blanco et al., 2007; Chaiswing et al., 2012). Here, we will challenge this concept, because we consider it an untenable simplification that ignores kinetic constrains and detracts the attention from more important, though more complex, catalytic events. The focus of this article is the importance of reaction kinetics vs. thermodynamics in the redox regulation of biological systems.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) Biol Chem 378: 827-836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 11 fermentation broth of A(600)2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b( + )/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k(cat) of 1.7 s(-1) and limiting Km values of 51.8 microM and 1.7 microM for t-butyl hydroperoxide and CfTXN2H6, respectively.

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.

Thiol-dependent hydroperoxide metabolism in parasites is reviewed in respect to potential therapeutic strategies. The hydroperoxide metabolism of Crithidia fasciculata has been characterized to comprise a cascade of three enzymes, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase, plus two supportive enzymes to synthesize the redox mediator trypanothione from glutathione and spermidine. The essentiality of the system in respect to parasite vitality and virulence has been verified by genetic approaches. The system appears to be common to all genera of the Kinetoplastida. The terminal peroxidase of the system belongs to the protein family of peroxiredoxins which is also represented in Entamoeba and a variety of metazoan parasites. Plasmodial hydroperoxide metabolism displays similarities to the mammalian system in comprising glutathione biosynthesis, glutathione reductase, and at least one glutathione peroxidase homolog having the active site selenocysteine replaced by cysteine. Nothing precise is known about the antioxidant defence systems of Giardia, Toxoplasma, and Trichomonas species. Also, the role of ovothiols and mycothiols reportedly present in several parasites remains to be established. Scrutinizing known enzymes of parasitic antioxidant defence for suitability as drug targets leaves only those of the trypanosomatid system as directly or indirectly validated. By generally accepted criteria of target selection and feasibility considerations tryparedoxin and tryparedoxin peroxidase can at present be rated as the most appealing target structures for the development of antiparasitic drugs.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or β-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or β-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.