In 1973, two major discoveries changed the face of selenium chemistry: the identification of the first mammal selenoenzyme, glutathione peroxidase 1, and the discovery of the synthetic utility of the so-called selenoxide elimination. While the chemical mechanism behind the catalytic activity of glutathione peroxidases appears to be mostly unveiled, little is known about the mechanisms of other selenoproteins and, for some of them, even the function lies in the dark. In chemistry, the capacity of organoselenides of catalyzing hydrogen peroxide activation for the practical manipulation of organic functional groups has been largely explored, and some mechanistic details have been clearly elucidated. As a paradox, despite the long-standing experience in the field, the nature of the active oxidant in various reactions still remains matter of debate. While many successes characterize these fields, the pharmacological use of organoselenides still lacks any true application, and while some organoselenides were found to be non-toxic and safe to use, to date no therapeutically approved use was granted. In this review, some fundamental and chronologically aligned topics spanning organoselenium biochemistry, chemistry and pharmacology are discussed, focusing on the current mechanistic picture describing their activity as either bioactive compounds or catalysts.

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 KM nor any substrate saturation. More recently, the reaction mechanism has been investigated by the density functional theory and nuclear magnetic resonance of model compounds mimicking the reaction cycle. The resulting concept sees a selenolate oxidized to a selenenic acid. This very fast reaction results from a concerted dual attack on the hydroperoxide bond, a nucleophilic one by the selenolate and an electrophilic one by a proton that is unstably bound in the reaction center. Postulated intermediates have been identified either in the native enzymes or in model compounds.

The Front Cover shows two lorries along the road directed to ‘H2O2 reduction’ destination. The selenol (SeH) lorry is in front, being faster than the thiol (SH) lorry. This cartoon represents the situation of glutathione peroxidase, in which the presence of selenium rather than sulfur warrants a significantly faster hydroperoxide reduction along the same mechanistic path. This can be explained on the basis of the chemistry of the two chalcogens as illustrated in our paper. More information can be found in the Full Paper by L. Orian and co-workers.

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(N)2 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.

Among the chalcogens, selenium is the key element for catalyzed H2O2 reduction. In organic synthesis, catalytic amounts of organo mono- and di-selenides are largely used in different classes of oxidations, in which H2O2 alone is poorly efficient. Biological hydroperoxide metabolism is dominated by peroxidases and thioredoxin reductases, which balance hydroperoxide challenge and contribute to redox regulation. When their selenocysteine is replaced by cysteine, the cellular antioxidant defense system is impaired. Finally, classes of organoselenides have been synthesized with the aim of mimicking the biological strategy of glutathione peroxidases, but their therapeutic application has so far been limited. Moreover, their therapeutic use may be doubted, because H2O2 is not only toxic but also serves as an important messenger. Therefore, over-optimization of H2O2 reduction may lead to unexpected disturbances of metabolic regulation. Common to all these systems is the nucleophilic attack of selenium to one oxygen of the peroxide bond promoting its disruption. In this contribution, we revisit selected examples from chemistry and biology, and, by using results from accurate quantum mechanical modelling, we provide an accurate unified picture of selenium's capacity of reducing hydroperoxides. There is clear evidence that the selenoenzymes remain superior in terms of catalytic efficiency.

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 hydroperoxides, 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 (more commonly named nitric oxide) 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.

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 kappa B 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.

Glutathione (GSH) plays a highly complex role in eicosanoid metabolism being a cofactor of terminal prostaglandin synthases and a substrate for leukotriene synthases, modulating enzyme activities, contributing to transcriptional activation, silencing key enzymes such as cyclooxygenases and lipoxygenases, as well as protecting related enzymes from oxidative inactivation. In human polymorphonuclear leukocytes, deprivation of GSH or the decrease of glutathione peroxidase activity by selenium deprivation dramatically increased 5-lipoxygenase activity. The sculpting of lipid peroxidation products to specific physiological mediators during evolution involved recruitment of GSH-dependent processes at various stages. The membrane-associated proteins in eicosanoid and glutathione metabolism family to which microsomal glutathione transferase 1 belongs, contains six members. Hematopoietic prostaglandin D synthase is a GSH-dependent enzyme belonging to the cytosolic glutathione transferase Sigma family. Lipocalin-type Prostaglandin D synthase is not strictly dependent on GSH, accepting various other thiols. GSH concentrations in cells can exhibit diurnal variation as well as vary across the cell cycle.

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.

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.

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.

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.

Glutathione peroxidases (GPxs) are enzymes working with either selenium or sulfur catalysis. They adopted diverse functions ranging from detoxification of H(2)O(2) 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 β-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.