The authors were asked by the Editors of ACS Chemical Biology to write an article titled "Why Nature Chose Selenium" for the occasion of the upcoming bicentennial of the discovery of selenium by the Swedish chemist Jöns Jacob Berzelius in 1817 and styled after the famous work of Frank Westheimer on the biological chemistry of phosphate [Westheimer, F. H. (1987) Why Nature Chose Phosphates, Science 235, 1173-1178]. This work gives a history of the important discoveries of the biological processes that selenium participates in, and a point-by-point comparison of the chemistry of selenium with the atom it replaces in biology, sulfur. This analysis shows that redox chemistry is the largest chemical difference between the two chalcogens. This difference is very large for both one-electron and two-electron redox reactions. Much of this difference is due to the inability of selenium to form π bonds of all types. The outer valence electrons of selenium are also more loosely held than those of sulfur. As a result, selenium is a better nucleophile and will react with reactive oxygen species faster than sulfur, but the resulting lack of π-bond character in the Se-O bond means that the Se-oxide can be much more readily reduced in comparison to S-oxides. The combination of these properties means that replacement of sulfur with selenium in nature results in a selenium-containing biomolecule that resists permanent oxidation. Multiple examples of this gain of function behavior from the literature are discussed.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

L-Selenocysteine (Sec or U) has been called the "21st amino acid". 1 Like the twenty common amino acids, selenocysteine is inserted during the translation of mRNA and has its own tRNA Sec and codon, UGA. This codon also serves as the opal stop codon. Decoding a UGA codon as one for selenocysteine requires a special structure in the 3′ untranslated region of the mRNA called a selenocysteine insertion sequence (SECIS) element. Because eukaryotic and prokaryotic cells use a different SECIS element to decode UGA as selenocysteine, the production of eukaryotic selenocysteine-containing proteins in prokaryotes is problematic. 2 Here, we describe a general semisynthetic route to proteins containing selenocysteine. 3,4 In "native chemical ligation", the thiolate of an N-terminal cysteine residue in one peptide attacks a C-terminal thioester in another peptide to produce, ultimately, an amide bond between the two peptides (Scheme 1). 5 "Expressed protein ligation" is an extension in which the C-terminal thioester is produced by using recombinant DNA (rDNA) technology. 6 We reasoned that selenocysteine, like cysteine, could effect both native chemical ligation and expressed protein ligation, and thereby provide a means to incorporate selenocysteine into proteins.We used AcGlySCH 2 C(O)NHCH 3 as a model thioester to test the feasibility of using selenocysteine in native chemical ligation. 7 Reaction with cystine ((CysOH) 2 ) in the presence of the reducing agent tris-(2-carboxyethyl)phosphine (TCEP) produced AcGlyCysOH, as well as some (AcGlyCysOH) 2 . When selenocystine ((SecOH) 2 ) was used in the same reaction, the product was (AcGlySecOH) 2 . 8 A selenolate (RSe -) is more nucleophilic than is its analogous thiolate (RS -). 9 Moreover, the pK a of a selenol (RSeH) is lower than that of its analogous thiol (RSH). 9a,10 These properties suggested to us that native chemical ligation with selenocysteine could be more rapid than with cysteine, especially at low pH. To test this hypothesis, we used the chromogenic thioester AcGly-SC 6 H 4 -p-NO 2 (1; Scheme 2) to determine the rate of native chemical ligation as a function of pH. 11 The resulting pH-rate profile is shown in Figure 1. Reaction with selenocysteine is 10 3 -fold faster than with cysteine at pH 5.0. Thus, native chemical ligation with selenocysteine can be chemoselective. 12 Having demonstrated the effectiveness of selenocysteine in native chemical ligation, we next set out to explore its utility in expressed protein ligation. As a model protein, we chose ribonuclease A (RNase A; EC 3.1.27.5; Figure 2), which has been the object of much seminal work in protein chemistry. 14 RNase A has 8 cysteine residues that form 4 disulfide bonds in the native For other means to incorporate selenocysteine into semisynthetic and synthetic proteins, see: (a) Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1989, 111, 4513-4514. (b) Fiori, S.; Pegoraro, S.; Rudolph-Bohner, S.; Cramer, J.; Moroder, L. Biopolymers 2000, 53, 550-564. (4) For a means to produce selenopeptide libraries o...

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.

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Thioredoxin reductase and thioredoxin constitute the cellular thioredoxin system, which provides reducing equivalents to numerous intracellular target disulfides. Mammalian thioredoxin reductase contains the rare amino acid selenocysteine. Known as the "21st" amino acid, selenocysteine is inserted into proteins by recoding UGA stop codons. Some model eukaryotic organisms lack the ability to insert selenocysteine, and prokaryotes have a recoding apparatus different from that of eukaryotes, thus making heterologous expression of mammalian selenoproteins difficult. Here, we present a semisynthetic method for preparing mammalian thioredoxin reductase. This method produces the first 487 amino acids of mouse thioredoxin reductase-3 as an intein fusion protein in Escherichia coli cells. The missing C-terminal tripeptide containing selenocysteine is then ligated to the thioester-tagged protein by expressed protein ligation. The semisynthetic version of thioredoxin reductase that we produce in this manner has k(cat) values ranging from 1500 to 2220 min(-)(1) toward thioredoxin and has strong peroxidase activity, indicating a functional form of the enzyme. We produced the semisynthetic thioredoxin reductase with a total yield of 24 mg from 6 L of E. coli culture (4 mg/L). This method allows production of a fully functional, semisynthetic selenoenzyme that is amenable to structure-function studies. A second semisynthetic system is also reported that makes use of peptide complementation to produce a partially active enzyme. The results of our peptide complementation studies reveal that a tetrapeptide that cannot ligate to the enzyme (Ac-Gly-Cys-Sec-Gly) can form a noncovalent complex with the truncated enzyme to form a weak complex. This noncovalent peptide-enzyme complex has 350-500-fold lower activity than the semisynthetic enzyme produced by peptide ligation.

Significance: Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. Recent Advances: The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. Critical Issues: We discuss in detail three major aspects associated with thiol/ disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. Future Directions: It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox active tetrapeptide of sequence Gly-Cys1-Sec2-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are: protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulsin, HIV Tat protein, H2O2, lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys2-mutant enzyme or the N- terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175- to 550-fold reduction in kcat. Our data supports the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue, but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates: (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, (iii) or are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC50 for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83 nM to 142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.