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Why Nature Chose Selenium Hans J. Reich, and Robert J. Hondal ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00031 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 8, 2016

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ACS Chemical Biology

1 WHY NATURE CHOSE SELENIUM By Hans J. Reich≠* and Robert J. Hondal‡* ‡

University of Vermont, Department of Biochemistry, 89 Beaumont Ave, Given Laboratory, Room B413, Burlington, VT 05405

≠

University of Wisconsin-Madison, Department of Chemistry, 1101 University Avenue, Madison, WI 53706

*

To whom correspondence should be addressed. Department of Biochemistry, University of Vermont, College of Medicine. 89 Beaumont Ave, Given Laboratory, Room B413, Burlington, VT 05405. Tel: 802-656-8282. FAX: 802-862-8220. E-mail: [email protected]

*

To whom correspondence should be addressed. Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706. Tel: (608) 262-5794 E-mail: [email protected]

ACS Paragon Plus Environment


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2 ABSTRACT 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 the inability of selenium to form pi-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 pi-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.


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3 Preface: The authors were asked by the Editors of ACS Chemical Biology to write an article titled “Why Nature Chose Selenium”, styled after the famous work of Frank Westheimer titled “Why Nature Chose Phosphates” (1). While Westheimer’s elegant chemical explanations for the use of phosphate in biology have found broad acceptance, currently the chemical reasons for the use of selenium in biology remain elusive and not widely agreed upon (2-16). This work is written for the occasion of the upcoming bicentennial of the discovery of selenium by the Swedish chemist Jöns Jacob Berzelius in 1817. We hope readers of this review on the chemistry of the “mysterious moon metal” (17) will be illuminated by our views. Discovery of Selenium: Oldfield describes the discovery of selenium by Berzelius as “Serendipity”, because he claims it was discovered during an investigation into an illness of the workers in a chemical factory at Gripsholm, Sweden (in part owned by Berzelius) that produced acetic, nitric, and sulfuric acids. As related by Oldfield, this illness was precipitated when the factory switched to a new, local source of sulfur ore (18). As the story goes, Berzelius thought this illness might be due to arsenic contamination of this sulfur ore, and the analysis of this ore led to the isolation of a new element (selenium). This story may be apocryphal as it is not mentioned by Trofast, who has reported on the discovery of selenium from a careful study of Berzelius’ original notes (19). Trofast reports that Berzelius declared, “…I, to mark its akin properties with tellurium, have named selenium, from Σελήνη, moon (goddess). What is more, it is in this regard, midway between sulfur and tellurium, and has almost more characters of sulfur than of tellurium.” Berzelius was a proponent of the theory of “electrochemical dualism” (20), which was a theory about the chemical nature of compounds. This theory held that all chemical compounds were held together due to neutralization of opposite electrical charges, as does occur in ionic compounds. It is tempting to think that the naming of selenium was a type of homage to this theory as tellurium had been named after Tellus, the Latin goddess of the Earth (Earth Mother). Ultimately, electrochemical dualism could not describe all types of chemical bonding and fell out of favor as a theory, but Berzelius’ discovery of selenium remains as a significant achievement in chemistry. Early Studies of Selenium in Biology: The first recognized role of selenium in biology was as a toxin. The investigation into the cause of “alkali disease” and “blind staggers”, diseases of livestock in the American West and Plains States by Kurt Franke and others, showed that these diseases were forms of selenosis due to the ingestion of high doses of selenium found in cereal crops, animal forage, and selenium accumulator plants such as Astragalus (known commonly as “locoweed”) grown in soils with high selenium content (21-24). It is remarkable that Franke at a very early date was able to show that the toxic form of selenium in locally grown grains was in the protein fraction of sulfuric acid hydrosylates. His experiments showed that selenium was “adsorbed on the protein molecule” (25). He was able to conclude that: “There is evidence that most of the selenium is in a compound very similar to cystine.” (26). Franke’s prescience that selenium would replace the sulfur atom of an amino acid is little recognized (26) and predates the discovery of the “21st” amino acid (27, 28), selenocysteine, by Thressa Stadtman (29) by 40 years! It should be noted that while blind staggers is often attributed to selenosis, it may in fact be caused by sulfate-related polioencephalomalacia due to contamination of water sources by sodium sulfate and magnesium sulfate (30).



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4 Selenium – Toxic and Essential: Selenium, like the moon, has two faces (31), as it is both toxic to all organisms, and essential to many bacteria and animal species. The essentiality of selenium to bacteria was to be discovered by Pinsent, who found that selenium was necessary for the activity of E. coli formate dehydrogenase in 1954 (32). A few years later selenium was discovered to be essential to animals independently by Patterson (33) and Schwarz (34, 35). Karl Schwarz, who even earlier was studying dietary liver necrosis in rats, had found that addition of methionine, vitamin E, or a “third factor” to the diet could prevent this condition (36). It is the identification of this “third factor” that Schwarz would become remembered for. Schwarz moved from Germany to the United States and took a position at the National Institutes of Health investigating the cause of exudative diathesis in chicks and liver necrosis in rats, diseases that were precipitated by a diet of torula yeast. Torula yeast is low in vitamin E, selenium, and sulfur amino acids, but rich in unsaturated fatty acids (37). These diseases did not occur if American brewer’s yeast (S. cerevisiae) was used instead. Schwarz was working on identifying the missing factor found in brewer’s yeast that prevented these diseases (38). Schwarz initially thought that this missing factor might be a vitamin, but experiments showed that the missing substance must be an inorganic compound. One of three elements, arsenic, selenium, and tellurium were suspected as the missing nutritional factor (38). Schwarz isolated the missing factor from acid hydrolysates of protein and called it “Factor 3” because it was the third substance identified that could prevent dietary liver necrosis (35). Jukes relates the story that Schwarz was able to identify selenium as “Factor 3”, the nutrient needed to prevent liver necrosis in rats, after Dr. DeWitt Stetten, then an Associate Director of the National Institute of General Medical Sciences, walked into Schwarz’s laboratory and smelled the distinct odor of a selenium-containing compound emanating from open test tubes of “Factor 3” in his laboratory (38). The odor may have been from dimethyl diselenide, which has a very sharp odor and is a decomposition product of selenomethionine. The two faces of selenium, essential and toxic, are unique in that the range between the amounts needed to maintain health or cause toxicity is quite narrow. The U. S. Department of Agriculture has a R.D.A. of 55 µg/day for adults (39), while the World Health Organization has established a toxic limit of 800 µg/day for adults (40). For this reason, Jukes refers to selenium as the “essential poison” (41). Diseases of Selenium Deficiency: Besides exudative diathesis and liver necrosis, selenium deficiency results in a number of other diseases of animals and humans (42). These include: white muscle disease (a muscular dystrophy disease mainly of sheep), mulberry heart disease, a disease affecting animal livestock and is so named due to hemorrhage of the heart that gives the organ the color and appearance of a mulberry (42), and in humans Keshan Disease (43-46) and Kashin-Beck Disease (47). Keshan Disease is a type of cardiomyopathy and may have an underlying viral etiology that is associated with selenium deficiency (48, 49). Kashin-Beck Disease is an osteoarticular disorder that resembles rheumatoid arthritis in some respects, but is much more severe. The beginning stages of the disease may involve destruction of the cartilage of the joints. The exact underlying cause of the disease is not known with certainty, but the disease is strongly associated with both selenium and iodine deficiency (50). It was the discovery of Keshan Disease and mammalian selenium-containing proteins that established selenium as an essential trace element for humans (45, 46, 51-53).


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5 Connection with Vitamin E: Although it has been shown independently by McCoy and Thompson that there is a biochemical function of selenium that must be distinct from that of vitamin E (37, 54), the presence of vitamin E can prevent, or attenuate, various animal diseases that are associated with selenium deficiency (55-68). This implies that at least one biochemical function of selenium is strongly connected with that of vitamin E. With the discovery of glutathione peroxidase as a selenoenzyme (52, 53), it became clear that one common function of the two is protection against lipid peroxidation (68). Another biochemical connection between selenium and vitamin E is vitamin C (ascorbic acid). The reduction of dehydroascorbic acid to ascorbic acid is catalyzed by thioredoxin reductase, a selenoenzyme (69). Ascorbic acid in turn can reduce the vitamin E radical formed in lipid bilayers after quenching a radical species. It is interesting to note that the coxsackievirus implicated as the underlying cause of Keshan Disease mutates to a more virulent form when the host is deficient in either selenium or vitamin E (70). This result could mean that both nutrients protect the host DNA from a mutation associated with an oxidation event, which leads to a more virulent form of the virus. Alternatively, Loscalzo has offered a possible mechanism that does not involve mutation of the virus; Low levels of glutathione peroxidase expression due to selenium deficiency, can result in oxidative stress that leads to myocardial injury and ventricular dysfunction, which in turn leads to cardiomyopathy characteristic of Keshan Disease (71). Selenium-Cancer Hypothesis: There has been a great deal of interest in the area of cancer chemoprevention by selenium since the late 1960’s. The earliest report of a relationship between selenium and cancer was by Nelson and coworkers (72) who reported that high dietary intake of selenium caused liver tumors in rats. However, experiments conducted later in the same decade also on rats showed that low doses of sodium selenite (Na2SeO3) protected against tumors induced by injection with dimethylaminoazobenzene (a 50% reduction in tumor incidence was reported) (73). Then in 1966, Shamberger and Rudolph showed that sodium selenide (Na2Se2) applied in a topical solution greatly reduced (730-fold) tumor formation in an induced mouse skin tumor model compared to DL-α-tocopherol (74). To help resolve the question of whether or not low levels of selenium were carcinogenic or not, the National Cancer Institute funded several studies that showed that selenium in the diet up to 8 ppm did not induce tumorigenesis (75-77), although there was still fear about the subject of selenium toxicity amongst the general public and some in the scientific community even after these studies (78). In a very influential letter to the editor of the Canadian Medical Association Journal, Shamberger and Frost hypothesized that “If selenium had an effect on public health, areas adequate or deficient could be expected to show different disease incidences or death rates.” (79). They pointed to a then recent study by Kubota and coworkers who had constructed a forage crop map of the U.S. that indicated which areas of the country had high or low selenium (80). Using this data they showed a correlation between areas of the U.S. with low forage crop selenium and a higher death rate. They also highlighted a study by Allaway and coworkers who had measured plasma selenium levels in multiple cities and counties in the U.S. and these measured low plasma selenium levels were correlated with higher cancer death rates (81). These ideas prompted Schrauzer to do a global study that examined the relationship between dietary selenium intake (they also measured plasma selenium) and cancer. He found inverse correlations for cancers of the large intestine, rectum, prostate, breast, ovary, and the lung (82). With respect to deficiency of selenium in soils, it should be noted that this is such a concern in Finland that the government has mandated the inclusion of selenium in fertilizer for agricultural land (83). To



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6 combat Keshan Disease in low selenium areas of China, Chinese health officials began adding sodium selenite to table salt (45). In the decades since 1970, numerous epidemiological, selenium supplementation studies, and clinical trials mostly supported the link between low selenium intake and a higher incidence of cancer (termed the selenium-cancer hypothesis). There are far too many examples of these kinds of studies to give a complete listing here, but some important ones are given in the reference list (84-94). Stampfer, Clark, Combs, Schrauzer, and Ip, give good summaries of the issues surrounding selenium and chemoprevention as well as a review of some of the important work done in this area (95-99). The selenium-cancer hypothesis perhaps reached its zenith in 1996 with a study led by Clark and Combs that showed supplementation with 200 µg/day of selenium in the form of selenized yeast led to significant reductions in colon, prostate, and lung cancers in a multicenter, double-blind, randomized, placebo-controlled cancer prevention trial (100). Notably, a KaplanMeier curve showed that selenium supplementation resulted in significant reductions in total cancer mortality (i.e. increased survival probability) over a 10 year time period (100). In response to this very positive outcome, the National Institutes of Health undertook an extremely large (35,533 men) randomized, placebo-controlled selenium supplementation trial. This trial was named the Selenium and Vitamin E Cancer Prevention Trial (SELECT) (101). An important distinction between the earlier trial and the SELECT study was the use of 200 µg/day of Lselenomethionine as the source of selenium instead of selenized yeast. The study found that there were no significant differences in any of the cancer endpoints. In other words, they did not find evidence that supplementation with selenium offered any protection against cancer (102). This result was extremely disappointing (to say the least) and contrary to many previous studies that supported the selenium-cancer hypothesis. Hatfield and Gladyshev have discussed some of the reasons for the large difference in experimental outcomes between the previous work (especially the work by Clark and coworkers) and the SELECT study (103). They note three significant differences: (i) The study by Clark (100) was initially undertaken to examine the effect of selenium supplementation for those at risk for skin cancer and so only considered risk factors for skin cancer during the randomization of subjects. (ii) The SELECT study used a different form of selenium, selenomethionine, while the study by Clark used selenized yeast. While selenomethionine can be used to make seleniumcontaining proteins, other forms of selenium could be important for chemoprevention of cancer. (iii) Last, participants in the SELECT trial had higher initial plasma levels of selenium than those in the study by Clark. This last fact suggests that supranutritional dietary selenium does not provide cancer protection, though epidemiology indicates that selenium deficiency can increase cancer incidence (vide supra). One seemingly contradictory fact about selenium and cancer is that overexpression of multiple selenoproteins such as glutathione peroxidase-2, Sep15, and thioredoxin reductase may help to promote cancer growth once the tumor has taken hold. (103-105). The fact that increased expression of a selenoenzyme such as thioredoxin reductase might help support tumor growth highlights an interesting fact about cancer cells and selenium. Cancer cells produce more reactive oxygen species (ROS) than normal cells and are adapted to a higher level of endogenously produced oxidants (106, 107). Thioredoxin and thioredoxin reductase are overexpressed in many human cancer types (108, 109), and this important selenium-containing antioxidant system helps to counteract oxidative stress experienced by cancer cells and enables cancer cells to resist


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7 programmed cell death (apoptosis). This fact contradicts the original idea of why selenium might help to prevent cancer; selenium as part of antioxidant enzymes, helps to prevent oxidative damage to DNA by free radicals and ROS. However, selenium can be involved in killing cancer cells using the opposite mechanism. Selenolates can react with molecular oxygen to produce superoxide (110) and the superoxide may push the cancer cell over an “oxidative cliff” from which the cell cannot recover, causing it to undergo apoptosis (107, 111). Indeed, there are clinical trials currently being undertaken to treat cancer that take advantage of this chemical reaction with selenium using sodium selenite (112, 113). Selenite and methaneseleninic acid, common forms of selenium in biology, are also very good oxidants. Both can oxidize thiol groups of enzymes, which could help push cancer cells towards apoptosis. Here we see two more “faces” of selenium, that as an antioxidant and an oxidant. Forms of Selenium in Biology: A very nice short review of the subjects discussed above can be found in (114). We now turn to the chemical forms of selenium used in biology and the types of chemistry selenium can perform. There are multiple chemical forms of selenium used in biology. Eight of these forms are shown in Figure 1. The principal form is that of selenocysteine, the 21st amino acid in the genetic code where it is co-translationally inserted into the polypeptide chains of selenoproteins (27, 28). In addition to being incorporated into proteins, selenium is found in nucleic acids, specifically as 5-methylaminomethyl-2-selenouridine (mnm5Se2U), where it is found in the wobble position of the anticodon loop in tRNAGlu, tRNALys, and tRNAGln in numerous species of bacteria (115-119). The selenolate of selenocysteine is a ligand for a number of coenzymes in bacteria such as in: (i) the molybdenum atom of molybdopterin guanine dinucleotide in formate dehydrogenase (120-124), (ii) the nickel atom of NiFeSe hydrogenases (125, 126), and (iii) iron in a putative iron-sulfur cluster in the methionine sulfoxide reductase from Metridium senile (127). Selenium is also found as the analog of ergothioneine in tuna, named as selenoneine (128). This novel selenium-biomolecule may be involved in mercury detoxification in fish (129). A methylated form is present in humans, but its function is not known (130). A very important dietary source of selenium is selenomethionine. Plants convert inorganic forms of selenium into selenomethionine, which is then converted into selenocysteine in animals via the trans-sulfuration pathway (reviewed in 131). Selenocysteine produced by this pathway is then converted into hydrogen selenide, which combines with ATP to produce selenophosphate (132-134) in a reaction catalyzed by selenophosphate synthetase (135-137). In bacteria (e. g. E. coli) selenophosphate is used as the nucleophile to attack the carbon-carbon double bond of dehydroalanyl-tRNA[Ser]Sec yielding selenocysteyl-tRNA[Ser]Sec (138). This specialized tRNA brings selenocysteine to the ribosome where it is incorporated into selenocysteine-containing proteins. Selenocysteyl-tRNA[Ser]Sec is also used to synthesize selenoproteins in eukaryotes, but a dehydroalanine-containing tRNA is not used as the acceptor for the attack by selenophosphate. Instead a phosphate group on O-phosphoseryl-tRNA[Ser]Sec is displaced by selenophosphate to produce selenocysteyl-tRNA[Ser]Sec (139, 140). Alliums such as garlic and onions tend to concentrate inorganic selenium in Se-methylselenocysteine (141) (not shown), which is converted into selenophosphate by a pathway that utilizes selenocysteine β-lyase and methaneselenol demethylase (142). Other forms of selenium in biology not shown in Figure 1 are: (i) selenocysteine as a ligand for the related molybdopterin–cytosine dinucleotide in carbon monoxide hydrogenase (143, 144), (ii) Se-methyl-N-acetylselenohexosamine, a selenosugar that is the major excretory



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Figure 1: Different chemical forms of selenium used in biomolecules. (1), selenocysteine (Sec, U), (2) 5-methylaminomethyl-2-selenouridine, (3) selenium, as selenocysteine, is a ligand for the molybdopterin guanine dinucleotide cofactor of formate dehydrogenase, (4) selenium, as selenocysteine, is a ligand for nickel in [NiFeSe] hydrogenases, (5) selenium, as selenocysteine, is a putative ligand for iron in an iron-sulfur cluster, and (6) selenium is found in selenoneine, the selenium analog of ergothioneine, (7) selenomethionine (SeMet), and (8) monoselenophosphate. selenium metabolite found in urine (145, 146), (iii) excretory compounds dimethyl selenide (breath) and trimethylselenonium (urine) (147, 148), (iv) selenite, which can react with glutathione to produce selenodiglutathione (149), and (vi) Se-methylselenocysteine, which in animals can be converted to methaneselenol by selenocysteine conjugate β-lyases (150, 151). A more complete list of biologically important selenocompounds can be found in (131). Selenium from methaneselenol can be converted into excretory forms dimethyl selenide and trimethyselenonium, or it can be converted into selenophosphate and put into selenoproteins (152). Ip and Ganther have compiled a considerable amount of data implicating methaneselenol as the form of selenium that is anti-carcinogenic (153-156). This may be due to redox cycling of this compound that induces apoptosis due to formation of superoxide (157). Why Selenium? – Clues from the Selenocysteine Insertion Machinery and Biogeochemistry: Selenocysteine, a major form of biological selenium, is a true proteinogenic amino acid because it meets the criteria met by the other 20 common amino acids: (i) it is encoded by DNA and it has its own unique codon (UGA), (ii) it has a unique tRNA that brings the aminoacylated selenocysteine residue to the ribosome, and (iii) is cotranslationally inserted into the polypeptide chain at the ribosome (158). The insertion of selenocysteine is much more


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9 complicated than cysteine and the other 19 proteinogenic amino acids as shown in Figure 2. The codon for selenocysteine is UGA, normally a stop codon. This UGA stop codon must be recoded as a sense codon for selenocysteine, and this recoding process requires an elaborate apparatus involving numerous accessory proteins and a special signal in the 3′-untranslated region of the mRNA of the selenoprotein (159-169). The details of the selenocysteine-insertion machinery are reviewed in (170-173). Second, it is extremely costly in terms of the cellular energy currency, ATP, to insert selenocysteine into a protein. It costs ~25 moles of ATP to insert 1 mole of cysteine into a protein (174). Given the multiple accessory proteins required to insert selenocysteine into a protein, the biosynthetic costs of a selenoprotein must be considerably more than that of a cysteine-containing protein. A third consideration for biology is the geological distribution of selenium in the Earth’s crust, as sulfur is much more abundant relative to selenium. This ratio is estimated to be as low as 6000:1 (175) and as high as 55,500:1 (176). In addition, selenium is not distributed evenly over the Earth’s crust (177). For example, there are both seleniferous and selenium deficient areas of China and the American west. Selenium deficient soils are especially consequential in China, New Zealand, and Finland (83). This means that animal life on land does not have equal access to this essential nutrient.

Figure 2: The eukaryotic Sec-insertion machinery. In eukaryotes, phosphoseryl-tRNASec kinase (PSTK) phosphorylates aminoacylated serine to form O-phosphoseryl-tRNA. Sep (Ophosphoserine) tRNA:Sec (selenocysteine) tRNA synthase, abbreviated as SepSecS, then converts O-phosphoseryl-tRNA to Sec-tRNA, using selenophosphate as the nucleophile to displace the phosphate group. Selenophosphate is produced by selenophosphate synthetase (SPS2). The Sec-tRNA is then bound by a special eukaryotic elongation factor (EFSec), and recruited to the ribosome at a UGA codon by the use of a special stem-loop structure in the 3′untranslated region of the mRNA (SECIS element) and a SECIS binding protein (SBP2). The details of Sec-insertion were first characterized in bacteria (161-165) and it should be noted that there are substantial differences in the Sec-insertion machineries of prokaryotes and eukaryotes (166-173). We also note that eukaryotes require additional accessory proteins for Sec-insertion not depicted here (172-173).



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10 Considering the three factors mentioned above it is natural to ask the question, “Why did Nature choose selenium?” The answer of the authors is that selenium must be able to perform some chemical function necessary for biology that sulfur is not very good at. In other words, there is a very large chemical difference between the two elements. If the chemical differences between selenium and sulfur were small, then Nature could abandon the use of selenium and not be dependent upon the factors listed above and use sulfur instead. The catalytic activity of the sulfur-containing enzyme may (or may not!) be lower than that of the selenium-containing ortholog, but Nature could compensate by making more of the sulfur version of the enzyme if needed. In the following sections, we review the chemical differences between sulfur and selenium. Selenium Containing Enzymes: While selenium is found in a variety of biomolecules as noted above, many of its important biological functions are due to its use in proteins and this is where our discussion will be focused. Most selenium-containing enzymes make use of the nucleophilic and reducing properties of the selenolate (Sec-Se–) form of a selenocysteine to perform redox reactions. After being oxidized, the resulting selenenic acid (Sec-SeOH) oxidation state is typically returned to selenolate by reduction with glutathione or a resolving Cys residue on the enzyme. The best studied selenoenzymes are: the glutathione peroxidases (Gpx), iodothyronine deiodinases (DIO, thioredoxin reductases (TrxR), and methionine sulfoxide reductases (Msr). There are eight Gpx isozymes in humans, five of which contain selenium (178), and they are an essential part of the system that scavenges hydroperoxides and hydrogen peroxide to prevent oxidative damage. There are three human Sec-containing iodothyronine deiodinases (179) found in the thyroid gland and other tissues, and they function to reduce the aryl iodide bonds of thyroxine (T4) and triiodothyronine (T3) to a C-H. There are three human Sec-containing thioredoxin reductases: a cystolic form, a mitochondrial form, and a specialized testes-specific enzyme (180). TrxRs help to maintain thiol-disulfide redox homeostasis via reduction of the small protein thioredoxin (Trx). Depending on the form of the enzyme, Msr reduces either free methionine sulfoxide or peptidyl methionine sulfoxide to methionine. There are four human Msrs, only one of which contains Sec (181). The Sec-containing Msr (MsrB1) is stereospecific for methionine-R-sulfoxide. The reduction of methionine-R-sulfoxide on actin promotes actin polymerization (182). Figure 3 shows the chemical processes shown to be, or likely to be, involved in the Sec-catalyzed reactions. In each case the oxidized selenium (Enz-Sec-SeOH, Enz-Sec-SeI or Enz-Sec-Se-SR) will be reduced back to Sec-Se– by GSH or another reductant in a reaction that involves nucleophilic attack at selenium. In each of these reactions the selenolate initially acts as a nucleophile (attacking an O-O, I-C, S-S or sulfinyl bond), the selenium of the formed selenenic acid derivative then acts as an electrophile, being attacked by a thiolate, and the catalytic cycle is completed by a reaction where selenolate behaves as a leaving group in the final reduction step of the catalytic cycle. It can be shown that selenium is likely to be more effective than the sulfur analog as a nucleophile, or as a leaving group, but not dramatically so. Typical Se/S rate ratios are one or two orders of magnitude (vide infra). It should be pointed out that while selenocysteine is found in the three major divisions of life, archeabacteria, eubacteria, and eukaryotes, there are entire classes of organisms that lack selenocysteine (Lepidoptera for example) where transformations such as the above are performed by cysteine (183, 184).


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Figure 3. Chemical processes mediated by Sec-containing enzymes (Enz-Se-: selenocysteine moiety of enzyme; GS–: glutathione; Trx: thioredoxin: B+-H: a proton donor). Chemical Property Comparisons Between Sulfur and Selenium: Even long before the interesting chemical and biological question “Why selenium?” was raised, comparison of the properties of sulfur compounds and their selenium analogs had drawn the attention of many chemists interested in selenium, often with the intent of identifying chemical properties that might make the selenium compounds useful. In many respects sulfur and selenium have very similar physical and chemical properties (10), they share all of the same oxidation states and functional group types (Figure 4), and their structures are often so similar that analogous



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12 compounds can easily co-crystallize. Here, we are interested in the significant chemical differences between the two chalcogens that might justify the metabolic cost of utilizing selenium. We will summarize some of those that have relevance to reactions of biological interest.

Figure 4: The structures and oxidation numbers of sulfur and selenium compounds that result from 2-electron oxidation reactions. Arrows show the conversion pathways between oxidation states. The open circle represents carbon and the number inside the circle represents the oxidation number of sulfur or selenium that it is attached to. The magenta X denotes a slow reaction. The [O] symbol represents a two-electron oxidant. Many of the significant differences between selenium and sulfur are a consequence of the usual changes on going from lighter to heavier elements. Heavier elements are more polarizable (“softer”) than lighter ones, and this usually leads to more rapid electrophilic and nucleophilic substitutions at the element. Most bond strengths to selenium are weaker than those to sulfur, and this results in substantially faster bond-breaking reactions. The weaker bonds to selenium mean that the sigma* orbital of the Se–X bond is lower in energy than that of a S–X bond, hence more reactive as an electron acceptor. Thus all oxidation states of selenium are much more electrophilic compared to sulfur analogs. It is also generally observed that higher oxidation states become relatively less stable for the heavier elements, and this is also true for selenium vs. sulfur. Heavier elements are also more tolerant of hypervalent bonding situations. The single most important use of selenium by synthetic organic chemists, the selenoxide elimination (Figure 5) to form alkenes, occurs at ca 100 ºC milder temperatures and ca 100,000 times as fast as related sulfoxide eliminations (185, 186).


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13

Figure 5. Relative rates of selenoxide (185) and sulfoxide (186) syn-eliminations. Acidity: The weaker bond to hydrogen, together with the increase in size and polarizability of the heavier atom, results in substantially lower basicity of selenolate versus thiolate by 3-4 pKa units (Figure 6) (187, 188). Thus cysteine is largely in the thiol state at neutral pH, whereas selenocysteine is almost fully ionized to a selenolate.

Figure 6. Comparison of selenol and thiol pKa values. Nucleophilicity: In spite of their lower basicity, selenolate ions are more nucleophilic by roughly one order of magnitude than thiolates, presumably a consequence of the higher polarizability of selenium (Figure 7). This is true for SN2 substitutions (189, 190) as well as for aromatic substitutions (191). In protic solvents the weaker hydrogen bond acceptor properties of selenolates vs thiolates contribute to higher nucleophilicity. Selenides are also more nucleophilic than sulfides (189).



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14

Figure 7. Comparison of selenium and sulfur nucleophilicities in SN2 substitutions using phenyl selenolate/thiolate (189), thioselenolate/thiocyanate (190), dimethyl selenide/sulfide with methyl iodide (189), and phenyl selenolate/thiolate with 4-nitro-1-bromofuran (191). Selenocysteine is also more nucleophilic then cysteine in a substitution at an acyl carbon (192). There is a much more significant difference in nucleophilicity between the two chalcogens at physiological pH, as selenols are completely converted to selenolates, whereas thiols are only slightly ionized. Since the selenolates/thiolates are almost certainly the active nucleophile, selenolates have the double advantage of both higher intrinsic nucleophilicity and a much higher fraction of the active form. In direct comparisons, this leads to ~2 orders of magnitude higher reactivity for selenolates versus thiols around pH 7. Some examples of this increased reactivity using selenocystamine (9) and cystamine (10) are shown in Figure 8. While Nature may take advantage of this difference in nucleophilicity at physiological pH, the pKa values of the nucleophilic cysteines in the context of a protein microenvironment can be greatly perturbed as evidenced by the pKa values of the active site Cys residues of papain, caricain, and ficin, which are 3.3, 2.9 and 2.5, respectively (193). As such, Nature may be able to increase the nucleophilicity of sulfur so as to minimize this difference at physiological pH (194), and it was concluded that selenium is not a “chemical necessity” in TrxR (195).


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15

Figure 8: Comparison of nucleophilic substitution by selenolate/thiolate on a disulfide and a peroxide (196). These reaction were done near neutral pH, where the cystamine (10) is largely in the SH form. When corrected to pH 10 (15), the relative rates are much smaller. Leaving Group Ability: Since selenolate is less basic than thiolate, selenolates are usually better leaving groups. As shown in Figure 9a, the selenol ester 11 (Y = Se) decomposes to ketene 180fold faster than the thiol ester 11-S (197), while in the biologically more relevant example in Figure 9b the selenosulfide (also referred to as a selenenylsulfide) is only 3-fold more reactive than the disulfide (196). This relatively small number may in part be due to stronger hydrogen bonding of the more basic thiolate at the transition state.



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16

Figure 9. Comparison of selenolate/thiolate leaving group abilities: (a) Ketene formation from an acyl selenide/sulfide. (b) Selenosulfide/disulfide exchange with thiolate as nucleophile. Hypervalency: One of the few bonding situations where Se–X bonds are stronger than S–X bonds occurs in hypervalent compounds, i.e. where the number of bonds plus lone pairs is greater than that allowed by the Lewis octet rule. Thus selenuranes (R4Se) form more easily than sulfuranes (R4S), and are much more stable. The same is true for the ate complexes R3Se– and R3S–. A computation of the energy of association (Me2Y + Me– → Me3Y–) is -0.3 kcal/mol for Y = S, and -13.1 kcal/mol for Y = Se (198). This effect has complex origins, but contributing factors are smaller steric effects due to the longer bonds to selenium, and the lower LUMO sigma* orbital which leads to a more favorable 3-center 4-electron hypervalent bonding situation. This greater tolerance for hypervalency (also shared by the neighboring element pairs Cl/Br and P/As, and which continues with the heavier elements Te, I and Sb) can be seen in many contexts. For example, the hypervalent sulfurane 12-S decomposes at -67 ºC with a halflife of 46 minutes, while the selenurane 12-Se has to be “heated” to 0 ºC before the decomposition rate is comparable, corresponding to a difference in activation energy of 2.9 kcal/mol (199). The addition product of chlorine and dialkyl sulfides (e.g. the Corey-Kim reagent (200)) is an ionic structure 13, whereas that of dialkyl selenides is covalent (14) (201). Another occurrence of stronger Se–X bonds occurs in bonding to metals. For example, Hg-Se bonds are stronger than Hg-S bonds (202).


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17 Electrophilicity: The greater tolerance for hypervalency of selenium has an important consequence, that nucleophilic attack on selenium (which typically forms or passes through hypervalent intermediates such as R4Se or R3Se-) usually occur much more rapidly than at sulfur, since the intermediate selenium compounds are lower in energy than sulfur analogs (203). Thus selenium compounds of all types are much better electrophiles than sulfur analogs and this can be seen in various contexts. For example bis(phenylthio)methane 15 is deprotonated with nBuLi, (204) whereas the selenium analog 16 is attacked at selenium (205).

In a closely related comparison, the rate of Ph/Tolyl exchange of diphenyl selenide (17Se) with tolyllithium is >2x104 times as fast as with diphenyl sulfide (17-S) (206).

Nucleophilic substitutions at a selenenyl halide such as 18-Se is substantially faster than at the analogous sulfenyl center, with kSe/kS ranging from 6000 for a dithiocarbamate, to 150 for cyanide (207). Similarly, nucleophilic attack of cyanide on PhSeSO2Ar is 5 orders of magnitude faster than on PhSSO2Ar (208).

Combination of nucleophilicity, electrophilicity, and leaving group ability: In a very careful study of the pH-dependence of the degenerate exchange of cystamine (10) with cysteamine and selenocystamine (9) with selenocysteamine, all three effects are operating in the same direction in the case of the selenolate/diselenide exchange reaction: the higher nucleophilicity of selenolate at physiological pH, the better leaving group ability of selenolate, and the stronger intrinsic electrophilicity of the center selenium atom. Using dynamic NMR techniques (line broadening for selenium, saturation transfer for sulfur), the selenolate/diselenide exchange reaction was measured to be 107 times a fast that of the thiol/disulfide exchange reaction at pH 7 (209). Weaker π-bonding: The larger size of selenium compared to sulfur (atomic radius of 115 pm compared to 100 pm for sulfur) results in larger hybridized orbitals, and this plus the longer bond



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18 length leads to weaker π-overlap. One consequence of this is that selenoesters are much less stabilized than thioesters by resonance with the carbonyl group (210). As shown in Figure 10, the acyl transfer between selenocystamine (9) and cystamine (10) strongly favors the thioester. Both Nature and chemists have taken advantage of the high reactivity of selenoesters as acyl transfer reagents. Selenoesters have been used to catalyze native chemical ligation reactions (211-213), and selenoprotein K uses its Sec residue to form a selenoester to catalyze acyltransfer of a palmitoyl group to a calcium channel (214).

Figure 10. Acyl-transfer equilibrium between a thiol ester and a selenol ester (210). Redox Properties: The greatest divergence between selenium and sulfur chemistry occurs in the redox reactions of the two elements. This is true for both 2-electron and 1-electron processes. We will begin our discussion with 2-electron oxidation reactions. One of the consequences of longer bond length of heavier elements is a reduced ability to form π-bonds of all types as introduced above. This means that even though a chalcogen-oxygen double bond is commonly depicted for convenience (as in Figure 4), a much better representation of the electronic structure of these compounds, particularly for selenium, is given in Figure 11.

Figure 11: Dipolar nature of chalcogen-oxygen bonds. Y = S, Se. Presumably because of stronger back donation of the lone-pair electrons on oxygen to acceptor orbitals (sigma* and possibly d orbitals) on sulfur compared to selenium, the Y-O dative bonds in selenoxides, selenones, seleninic acids, and selenonic acids are weaker for selenium, have substantially more dipolar character, and are relatively less favored than for sulfur. This can be seen in many property changes when S/Se comparisons are made. For example, alkyl selenones are excellent alkylating agents (215, 216), while sulfones are completely unreactive.


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19 The differences in electronic structure noted above in the chalcogen oxides leads to large differences in their chemical properties. For example, dimethyl selenoxide (pKa of conjugate acid 2.55 (217)) is substantially more basic than dimethyl sulfoxide (pKa of conjugate acid -1.54 (218)). Thus in an acid-catalyzed reaction at a given pH, selenoxides will have ca. 104 as high a concentration of the reactive protonated selenoxide compared to the protonated sulfoxide. This would already result in dramatically higher reactivity, but in addition the selenium bears more positive charge, and is inherently more electrophilic than sulfur, combining to give very much higher rates of nucleophilic attack at selenium. Another example of the enhanced reactivity of the Se-oxide relative to an S-oxide is the racemization of selenoxides compared to sulfoxides. The acid-catalyzed racemization of sulfoxides is a slow and difficult process. Selenoxides racemize many orders of magnitude faster than the corresponding sulfoxides. The data for the selenoxide inversion in Figure 12 was obtained from dynamic NMR measurements, whereas the sulfoxide data was obtained from the epimerization of one diastereomer to the other. The inversions proceed by different mechanisms; the selenoxide is first order in [H+] independent of whether HCl or H2SO4 was used, so it follows Path A. The sulfoxide, on the other hand was too slow to measure with H2SO4. With HCl as acid, the sulfoxide inversion rate can be measured, but is second order in HCl, so it proceeds by a mechanism than involves formation of R2SCl2 (Path B). A direct comparison of the rates was thus not possible. However, one can estimate that kSe/kS is minimally on the order of 1013 at pH 0, of which roughly 104 can be ascribed to the higher basicity of the selenoxides, and 109 to the higher electrophilicity of the protonated selenoxides towards attack by water (219).

Figure 12. Rates of acid-catalyzed inversion of sulfoxides and selenoxides in D2O at 25 °C. A common effect seen in most heavier-lighter element comparisons is a stronger preference for lower oxidation states in the heavier elements. Selenium is no exception. This effect can be seen directly in several contexts. For example, selenoxides are able to oxidize sulfides to sulfoxides (220). A semi-quantitative measure of this effect is provided by the [2,3]sigmatropic rearrangement equilibrium between allyl sulfoxides and selenoxides, where the



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20 equilibrium is slightly on the sulfoxide side for sulfur, and strongly on the selenenate ester for selenium. In the specific case shown in Figure 13, this shift in equilibrium was approximately 13 kcal/mole (the two equilibria are not strictly comparable since they were measured at different temperatures). This is one of the largest S/Se divergences reported (221).

Figure 13: Equilibrium constants between selenoxide-selenenate (-80 °C) and sulfoxidesulfenate in CD2Cl2 at -30 °C (221). Related to this is the divergent behavior of sulfur dioxide and selenium dioxide. SO2 is considered a mild reducing agent, whereas SeO2 is a mild oxidizing agent (Riley oxidation). Both reagents react with alkenes to form intermediate allylsulfinic and allylseleninic acids (Figure 14). However, the allylsulfinic acid simply reverts to the alkene and SO2 (222), maintaining the higher oxidation state at sulfur, whereas the allylseleninic acid undergoes [2,3]sigmatropic rearrangement to form the allyl ester of a divalent selenium species, which rapidly hydrolyzes to the allyl alcohol (223).


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21

Figure 14. The divergent behavior of SO2 and SeO2 towards alkenes. The difference in electronic structure of the chalcogen oxides also leads to different rates of oxidation and reduction, first introduced in Figure 4. Although the first oxidation of sulfides/selenides to sulfoxides/selenoxides is fairly comparable, with selenium being slightly more reactive, the second oxidation is very much more difficult for selenoxides. This is in part a consequence of the much higher dipolar character of the Se–O bond resulting in lower nucleophilicity of the lone pair on Se. In fact, oxidation of sulfides to sulfoxides requires a delicate touch to avoid over-oxidation to the sulfone because the second oxidation is only a little slower than the first. In comparison, any reasonable oxidant can be used with selenides, since the second oxidation to selenones is much slower. Another consequence of the higher dipolar character of selenoxides versus sulfoxides is that the lone pair on selenium is less nucleophilic than the lone pair on sulfur. Thus sodium benzenesulfinate anion alkylates on sulfur to give the sulfone 19 (this is a standard synthetic method for the synthesis of sulfones), whereas the seleninate anion alkylates on oxygen to give a seleninate ester 20 (224).

The behavior of these reagents with dienes is similarly enlightening as SO2 forms the cyclic sulfone 21, whereas SeO2 forms the cyclic seleninate ester 22 (225). It is believed that the SO2 reaction also proceeds through a cyclic sulfinate ester, but this rapidly rearranges to the more stable sulfone (226).



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22

The redox reactions of thiol/selenols follow a pattern similar to that of the sulfides/selenides. The redox potential of selenols is much lower compared to that of thiols (-381 mV with DTT as the reductant vs. -179 mV with glutathione as the reductant in the context of a glutaredoxin peptide fragment using either Sec or Cys) (227). This means that the equilibrium constant for the oxidation of a diselenol to a diselenide, greatly favors the diselenide. In comparison, the equilibrium constant for the dithiol/disulfide pair was 600-fold lower in this same experiment and favors the formation of the dithiol. A similar experiment conducted not on a peptide fragment, but in the context of a folded protein (glutaredoxin-3) showed that the ratio of equilibrium constants was 8500:1 in favor of the diselenide (228). This experimental system also showed that the rate of formation of the diselenide was much faster than the rate of formation of the disulfide. While the examples above show that incorporation of Sec in place of Cys results in a very large change in redox potential in the context of a peptide or protein microenviroment, Rozovsky and coworkers have shown that insertion of Sec does not necessarily confer a large change in redox potential in the context of a protein (229). They thusly note that the lower redox potential of a selenosulfide bond may not be the raison d'etre for the use of Sec. Oxidation of a selenol to a selenenic acid is presumably faster than the same oxidation of a thiol to a sulfenic acid, but there is very little data available for these compounds as they are unstable and undergo rapid disproportionation to diselenides and seleninic acids, or become further oxidized. There are some examples of the synthesis of stable sulfenic/selenenic acids that make use of bulky substituents to sterically crowd the chalcogen oxide to protect it from condensation/disproportionation (230-234), but there is only one report that makes a direct comparison of the chemical properties of the two so far as we are aware (233). A significant finding of this work was that the bond dissociation energy (BDE) of the O–H bond in a selenenic acid (81.2 kcal/mol) is higher than a Se–H bond (78.9 kcal/mol). Moreover, the opposite trend was reported for sulfur; the BDE for the O–H bond in a sulfenic acid was reported as 68.6 kcal/mol, which is weaker than a S–H bond (87.6 kcal/mol). This greater strength of the O–H bond in the selenenic acid led to a slower rate of reaction with a peroxyl radical compared to the sulfenic acid (233). Oxidation of selenenic/sulfenic acid results in formation of seleninic/sulfinic acids. The chemical comparison between seleninic and sulfinic acids is analogous to that between SeO2 and SO2, or between their hydrates, selenious acid (O=Se(OH)2) and sulfurous acid (O=S(OH)2). That is, seleninic acids are weak oxidizing agents (in fact seleninic anhydride has been proposed as a useful oxidant for many functional groups (235-237)), whereas sulfinic acids are weak


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23 reducing agents. In a striking example of this (Eq. 1), Kice reported that PhSeO2H and PhSO2H react with each other to reduce the former and oxidize the latter (Eq. 1) (238).

Seleninic acids are weaker acids than sulfinic acids by about 2 pKa units (PhSeO2H 4.79 (239), PhSO2H 2.76 (240)), a consequence of the higher electronegativity and better π-acceptor properties of sulfur versus selenium. No information on the basicity of seleninic acids could be found (pKa of PhSe(OH)2+). However, one can predict that they would be substantially more basic than sulfinic acids (in analogy with selenoxides and sulfoxides), and thus acid catalyzed substitutions at selenium would also be greatly accelerated. This is indeed what is observed. The reduction of seleninic acids by thiols is extremely fast and may be biochemically important (241-243). This reaction probably involves the sequence of steps outlined in Figure 15. Benzeneseleninic acid is reduced extremely rapidly by thiols, in one experiment, i-Pr-SH reacted with PhSeO2H in under 30 seconds at -90 °C (Reich, H. J., Kolonko, K. J., unpublished results), whereas PhSO2H does not detectably react with thiols in weeks at room temperature (243). The rate of reaction of the seleninic acid form of the synthetic selenoenzyme selenosubtilisin (244) with an aromatic thiol (3-carboxy-4-nitro-benzenethiol) gave an apparent second order rate constant of 1.8x104 M-1 s-1 (pH 5.0), while that of an alkaneseleninic acid was ~200-fold faster (3.3x106 M-1 s-1) (242). These fast rate constants mean the lifetime of a seleninic acid is on the order of seconds when the concentration of thiol is in the millimolar range. From such observations one can estimate that the rate of reaction of thiols with seleninic acids is at least 106 faster than their reaction with sulfinic acids.

Figure 15: Probable mechanism of the reduction of seleninic acids by thiols (245). As discussed above, the reduction of seleninic acid to the -2 and 0 oxidation states is very fast in comparison to the same reduction of sulfinic acids. This represents a point of large divergence in the chemical reactivity of the respective chalcogens. The rate of oxidation of seleninic acids to selenonic acids is also a point of divergence in comparison to the oxidation of



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24 sulfinic acids to sulfonic acids (noted by Kice (238)), with the latter being ~2200-fold faster. At pH 7.1 the rate of oxidation of benzenesulfinic acid is 2.7x10-3 M-1 s-1, while that of benzeneseleninic acid is 1.2x10-6 M-1 s-1 using H2O2 as the oxidant (243). One-electron redox reactions. Figure 16 shows a pair of reactions in which the one involving selenium is much slower than the sulfur analog. In a very significant paper, Koppenol, Nauser and coworkers showed that when a thiyl radical is formed in a peptide, Cα–H abstraction is greatly favored, while the same reaction of a selanyl radical is slow (15, 246). The significance of this will be discussed in the following section. The one-electron redox potential of the two chalcogens is also different; the RS•, RSH/H+ couple has a redox potential of 0.92 V, while the RSe•, RSeH/H+ couple has a potential of 0.43 V (246). This difference in redox potential means that a thiyl radical is capable of oxidizing tyrosine and tryptophan residues to form the corresponding amino acid radical, while the selanyl radical will not perform the same oxidation (246).

Figure 16. Comparison of the rates of intramolecular hydrogen abstraction by thiyl and selanyl radicals (246). Why Selenium? Rate Advantage versus Redox Advantage: A seemingly logical conclusion to the question of “Why selenium?” was arrived at relatively early in the field by replacement of the catalytic Sec residue of several Sec-containing enzymes with a Cys residue (247-250). The replacement of Se with S resulted in mutant enzymes with greatly impaired catalytic activity. Conversely, replacement of S with Se in Cys-containing enzymes resulted in enhanced catalytic activity (228, 251-253). Thus one answer to the question of “Why selenium?” is that the use of Se in oxidoreductases of the type discussed here provides a catalytic advantage to the enzyme. However, Stadtman made an observation that is often overlooked in the study of selenium in biology. She found that the catalytic activity of the Cys-containing selenophosphate synthetase from E. coli had higher specific activity than the Sec-containing enzyme from H. influenzae (254). She noted that: “These results taken together suggest a role for selenocysteine of H. influenzae that is not catalytic.” Kanzok and coworkers also concluded that the use of selenium was not required in the active site of TrxR because a Cys-ortholog enzyme had comparable activity toward its cognate substrate (195). If the use of Sec and other forms of selenium is not entirely related to enhanced catalytic function relative to sulfur, then a second possibility is related to selenium’s advantageous redox properties discussed above.


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25 Besides the gain of increased catalytic activity of most reaction reaction types upon Se for S substitution in enzymes, an additional gain of function that occurs is a gain of peroxidase activity (244, 255-260). This gain of function fits well with its superior redox properties relative to sulfur described above. The functional definition of a peroxidase is outlined in Figure 17. Selenium confers peroxidase activity to an enzyme because it is both a good nucleophile and a good electrophile. This property allows it to easily cycle between reduced and oxidized states without becoming permanently oxidized. In this respect, the redox properties of selenium more closely resemble a transition metal in comparison to sulfur. The gain of peroxidase activity conferred by selenium led to the hypothesis that selenium confers resistance to irreversible oxidation and inactivation due to the difference in redox chemistry between selenium and sulfur shown in Figure 4 (12, 263). This hypothesis is a late manifestation of an idea that has been expressed in other forms, or went unrecognized by researchers who did not realize the significance of their data. This hypothesis is reviewed below.

Figure 17: The peroxidase cycle for Sec- and Cys-peroxidases. Both types have a nucleophilic selenolate/thiolate that reacts with an oxidant to form a selenenic/sulfenic acid, which is then resolved by another thiol to form a mixed selenosulfide/disulfide. The active enzyme is regenerated by addition of a second mole of thiol. Addition of selenium to an enzyme imparts peroxidase activity because a selenolate is a good nucleophile, allowing for reaction with an oxidant, and the Se-oxide is an excellent electrophile that allows it to be attacked by a thiol to release water. In the mechanism, a selenolate is also a leaving group. Chemically, sulfur is less reactive with respect to all three of these properties. However, Cys-peroxidases can be highly efficient catalysts presumably because the protein microenvironment can tune the reactivity of sulfur to compensate for the absence of selenium using a combination of these properties (261). One large difference is the inability of the Cys-SO2– form of the Cys-peroxidases to return to the active catalytic pathway by reduction with exogenous thiol, while reduction of the Sec-SeO2– form is possible for Sec-peroxidases as shown by the work of Hilvert (242). Peroxiredoxin is one exception to this rule, but it requires a special repair enzyme to reduce the Cys-SO2– form of the enzyme (262), while the reduction of Sec-SeO2– of Sec-peroxidases can be easily reduced by ascorbate and glutathione. Chaudière and coworkers were the first to note that Sec may have evolved in an enzyme to prevent “self inactivation” (249). This observation was made by studying the Cys-mutant of Gpx1, which had much lower activity than the Sec-containing Gpx1, but was readily inactivated in the presence of its substrate, H2O2 and organic hydroperoxides. Chaudière hypothesized that the Cys-mutant Gpx1 was inactivated due to oxidation of the peroxidatic Cys residue to CysSO2– with possible β-elimination to form dehydroalanine. This hypothesis was perhaps validated several decades later by Bellelli and coworkers who crystallized the Cys-mutant of Sec-



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26 containing Gpx4 from Schistosoma mansoni (264). This X-ray crystal structure of the Gpx4 mutant showed the presence of a sulfonic acid residue (see Figure 18) (264). The Cys-mutant of S. mansoni Gpx4 was inactive at all stages of purification, even though reducing agents were present. One interpretation of this data that matches Chaudière’s observation is that the Cysmutant enzyme was able to react with the oxidant, but the S-oxide that formed was not electrophilic enough to be resolved back to the active form of the enzyme, and this results in overoxidation and inactivation of the Cys residue of the mutant enzyme.

A

B

Figure 18: (A) A close in view of the catalytic triad of S. mansoni Gpx4 consisting of Cys43 oxidized to a sulfonic acid, Gln78, and Trp132 (PDB 2v1m). (B) A close in view of the catalytic triad of selenosubtilisin showing Sec221 in the seleninic acid form along with Asp32 and His64 (PDB 1sel). The selenium atom is colored magenta. Very recently, Ursini, Flohé, and coworkers presented evidence that Sec-containing rat Gpx4 avoids overoxidation of the Sec residue by using a backbone nitrogen to attack the selenenic acid intermediate to form an 8-membered ring selenenamide when glutathione becomes limiting (265). Formation of the selenenamide is thought to be a type of safety mechanism that prevents overoxidation and inactivation of the Sec-Gpx. The analogous sulfenamide could not be detected in the Cys-mutant rat Gpx4 (265). Instead, the peroxidatic Cys residue of the mutant became irreversibly oxidized to the sulfonic acid form. The experimental evidence of selenenamide formation in the native protein fits well with earlier work by Reich and coworkers who synthesized model compound 23, which became oxidized to both the selenenamide 24 and seleninamide 25 in the presence of an oxidant (266). Selenenamides have the interesting property of being significantly more basic (3.4 pK units) than the corresponding sulfenamides (267). This property should greatly enhance opening of the ring by an attacking thiol. This is yet another large chemical difference between sulfur and selenium.


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27

This story continues in 1993 when Hilvert and coworkers discovered that selenosubtilisin, a semisynthetic selenoenzyme (244), was not inactivated by oxidation to the Enz-Sec-SeO2– form as the enzyme could regain activity by the addition of two additional moles of thiol (242). There is very strong evidence that selenosubtilisin is capable of existing in the Enz-Sec-SeO2– form as supported both by X-ray crystallography (268) (see Figure 18B) and 77 Se NMR spectroscopy (269). At the time, the importance of this discovery as it relates to irreversible oxidative inactivation was unclear since little was then known about the existence of the corresponding sulfinic acid form in proteins. This is not the only example of the seleninic acid form in an enzyme. Branlant and coworkers converted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into a selenoenzyme by producing the protein in a cysteine auxotroph and replacing cysteine in the media with selenocysteine (256). Replacement of Cys in GAPDH with Sec resulting in a gain of peroxidase activity, but more importantly they showed that the enzyme existed in the Enz-Sec-SeO2– form (by the use of mass spectrometry) without resulting in inactivation (256). It is known however, that oxidation of GAPDH to the Enz-Cys-SO2– form results in permanent oxidative inactivation (270). While these examples show that the seleninic acid form of a selenoenzyme can be part of a catalytic cycle, there is no evidence as of yet that a natural selenoenzyme uses this redox form. It is not widely appreciated that selenium is also found as a ligand for metal clusters in hydrogenases as shown in Figure 1. Early work on [NiFeSe]-hydrogenases showed that this class of enzymes was oxygen tolerant (271), unlike [Fe-Fe]-hydrogenases and most [NiFe]hydrogenases. Maroney and coworkers provided evidence that selenium confers oxygen tolerance in [NiFeSe]-hydrogenases by synthesizing nickel-containing mimics of the active site that had either sulfur or selenium ligands as shown in Figure 19 (272). Reaction of the thiolato complex with molecular oxygen resulted in a monosulfinato complex. In striking contrast, the selenolato complex resisted oxidation. In follow up work, Maroney comments that substitution of selenium for sulfur in the [NiFeSe]-hydrogenase from D. baculatum (a strict anaerobe) makes “…the enzyme more stable to oxidation and may be isolated in air in a state that does not require reductive activation.” (273). This view of selenium in an enzyme strongly diverges from the earliest view of the use of selenium in an enzyme expressed by Böck: ‘‘UGA was originally a sense codon for Sec in the anaerobic world, perhaps two to three billion years ago, and after introduction of oxygen into the biosphere this highly oxidizable amino acid could be maintained only in anaerobic organisms or in aerobic systems which evolved special protective mechanisms.’’(162). Based on the results of Maroney, it is tempting to speculate on the exact opposite scenario; The use of Sec in proteins reached a maximum during the oxygen peak of the Permian period and then declined as the amount of oxygen in the atmosphere declined.



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28

Figure 19: Reaction of thiolato or selenolato nickel complexes with molecular oxygen. Only the sulfanato complex resulted in oxidation (272). In a follow up to the work by Maroney more than a decade later, Armstrong and coworkers performed a study of the [NiFeSe]-hydrogenase from D. baculatum using protein film voltammetry (274). The goal of the study was to more completely characterize the A and B states of the enzyme, with the A state corresponding to an “Unready” form (formed upon reaction with O2), while the B state corresponds to the “Ready” form. The “Ready” and “Unready” states are so named because of their kinetic characteristics with respect to reactivation of the enzyme (slow and fast, respectively). An important finding of this work was that the enzyme was capable of hydrogenase activity in the presence of 1% O2, and the “Unready”, A-form of the enzyme was reactivated 100-fold faster compared to the non-selenium containing A-form of the [NiFe]hydrogenase from A. vinosum. Armstrong contemplated that the inactive A-form is due to the formation of a Se-oxide that is rapidly reduced to the active form of the enzyme (274). The role of selenium in conferring oxygen tolerance to [NiFeSe]-hydrogenases was further addressed by Matias and coworkers who solved the X-ray crystal structure of the [NiFeSe]-hydrogenase from D. vulgaris (275). The electron density map of the [NiFeSe] complex was interpreted in terms of three structures in a 70:15:15 ratio, respectively, as shown in Figure 20. Structures I and II were persulfurated, while the third had a direct Ni-Se linkage. Their analysis revealed that selenium in I and II shields the nickel atom from small molecules such as molecular oxygen, preventing inactivation from the formation of an oxo ligand at the nickel center. This may be due to selenium’s ability to form strong metal bonds, as it does with mercury (202). However the presence of the Sec residue in the enzyme does not prevent other oxidation events such as the conversion of Cys75 from a nickel sulfide to a nickel sulfinate.


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29

Figure 20: The three structures of the NiFe center in the [NiFeSe]-hydrogenase from D. vulgaris (275). Structure I corresponds to the oxidized form, while structure III corresponds to the reduced form. Structure II is an intermediate between I and III. Another example of selenium conferring resistance to oxidation comes from a Sec for Cys substitution in CYP119, a cytochrome P450 heme-containing monooxygenase from Sulfolobus acidocaldarius (276). Oxidation of SeCYP119 with excess m-chloroperbenzoic acid (mCPBA) led to the presence of a new oxidized species that absorbed at 406 nm in a magnetic circular dichroism spectrum. The identity of this oxidized species could not be elucidated, but this oxidized form could be reduced back to the original state in the presence of a reducing agent such as DTT. Importantly, the introduction of selenium into the enzyme protected against heme destruction compared to the protein containing sulfur (either selenium or sulfur are ligands for the iron center of the heme), where significantly more heme destruction occurred (Figure 21).

Figure 21: (left panel) Protection against heme destruction by the introduction of selenium into the P450 monooxygenase from S. acidocaldarius (276). (right panel) Heme destruction was pronounced in the cysteine-containing protein in the presence of excess mCPBA (indicated by dark, double arrows), but not so the selenium-containing version. An unidentified, oxidized form of Sec could be reduced back to the parent compound by addition of dithiothreitol (DTT) as indicated, but the oxidized form of Cys could not. The most recent evidence for the hypothesis that selenium is used to prevent irreversible oxidative inactivation comes from Hondal’s laboratory, who tested this hypothesis in Seccontaining thioredoxin reductase (TrxR). The approach was to compare the ability of the Seccontaining TrxR to resist oxidative inactivation compared to a Cys-ortholog from D. melanogaster (DmTrxR) (263). DmTrxR was used for direct mechanistic comparisons because it has similar enzyme architecture to the mammalian Sec-containing enzyme, but Cys replaces Sec (195, 277). DmTrxR is 50% inactivated at 1 mM H2O2, while the Sec-TrxR retained virtually all of its activity as shown in Figure 22. To support the hypothesis that selenium confers the ability to resist oxidative inactivation to an enzyme, Cys was replaced with Sec using a sophisticated protein engineering technique called expressed protein ligation (278), and then the ability of the



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30 mutant enzyme to resist H2O2-mediated inactivation was measured by assaying for the remaining activity. As shown in Figure 22, replacement of a single atom in the enzyme, selenium for sulfur, enabled resistance to oxidative inactivation by H2O2 (263), identical to the examples discussed above. Because of this effect, we call this the “Sec-rescue”-TrxR. Both the Sec-TrxR and the Sec-rescue-TrxR were also able to resist inactivation by one-electron oxidants such as hydroxyl radical as had been predicted by Steinmann and coworkers (15), but Cys-containing DmTrxR could not. Figure 22B: Depiction of the gain of function that Sec confers to an enzyme in the “SecRescue”-TrxR – resistance to oxidative inactivation. Figure 22A: (bar graph left) Activity of TrxR enzymes after H2O2 exposure. The blue bar is the activity of the Cys-TrxR (DmTrxR), the red bar is mouse Sec-TrxR, and the green bar is the CysSec mutant of DmTrxR. In this experiment, the enzymes were first exposed to H2O2. Then the H2O2 was quenched with catalase and substrate (Trx) was added and the amount of activity remaining was measured. Taken from (263). Conclusion: Almost all chemical reactions involving selenium are faster in comparison to the same reaction with sulfur. For this reason it is tempting to conclude that Nature chose selenium to replace sulfur for this enhanced chemical reactivity in order to accelerate enzymatic reactions. In contrast, our answer to this question is that Nature has chosen selenium due to its unique ability to react with oxygen and related ROS in a readily reversible manner. Both sulfur and selenium are good nucleophiles that react with ROS in 2-electron oxidation events, and in so doing become oxidized. The S-oxides and Se-oxides that are formed in this process show very strong divergence in their chemical reactivities, due in large part to very weak pi-bonding in the Se-oxide. As a result, Se-oxides have a much stronger ability to be rapidly reduced back to the original state in comparison to S-oxides. The ability of selenium to both rapidly become oxidized and then be rapidly reduced has been referred to as the “selenium paradox” (12). Evidence for this “selenium paradox” comes from the gain of function that selenium confers to both natural and artificial selenoenzymes; selenium confers resistance to inactivation by oxidation as the numerous examples discussed above show. Closely related to the reversibility of 2-electron oxidation events is the enhanced stability of the selenayl radical compared to the thiyl radical (15, 246). This means that seleniumcontaining proteins are much better able to withstand one-electron oxidation events (263). Both of these hypotheses fits well with the idea that oxygen in the biosphere is perhaps the strongest evolutionary force in the history of life on Earth (279) and the chemistry of selenium is ideally suited to respond to 1- and 2-electron oxidation events.


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31 ACKNOWLEDGMENT The authors would like to thank Michael Maroney of the University of Massachussetts-Amherst, Paul Copeland of Rutgers University, and Raymond F. Burk of Vanderbilt University (emeritus), for providing critical commentary and encourgement. The authors would also like to thank Stephen Everse of the University of Vermont and Francessco Angelucci of the University of L'Aquila for help in constructing figures. DEDICATION This paper is dedicated to Raymond F. Burk on the occasion of his recent retirement from Vanderbilt University and for his many years of dedication to the study of selenium biochemistry. GLOSSARY 1. selenocysteine: The selenium analog of cysteine. It is the 21st amino acid in the genetic code. 2. selenouracil: The selenium analog of thiouracil. Both thiouracil and selenouracil are modified bases found in the anticodon loop of tRNA molecules. 3. selenoxide elimination: A chemical method for generating carbon-carbon double bonds that involves abstract of a hydrogen that is beta to the selenoxide. This reaction is very fast and is perhaps the single most important use of selenium by synthetic organic chemists 4. selenenic acid: The selanyl monooxide form of selenium. It is the selenium analog of sulfenic acid. 5. seleninic acid: The selanyl dioxide form of selenium. It is the selenium analog of sulfinic acid. 6. selenonic acid: The selanyl trioxide form of selenium. It is the selenium analog of sulfonic acid. 7. selenoxide: The monooxidized form of a selenide. It is the selenium analog of a sulfoxide. 8. selenone: The dioxidized form of a selenide. It is the selenium analog of a sulfone. 9. selanyl radical: The radical formed on selenium when a selenol loses a hydrogen atom. It is the selenium analog of a thiyl radical. 10. oxidative inactivation: The process by which an enzyme loses activity due to oxidation of a functional group important for catalytic activity. Note to Editor on Figure Sizes: Figure 1: Should be a 1-column figure Figure 2: Should be a 1-column figure Figure 3: Should be a 1-column figure Figure 4: Should be a 2-column figure



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32 Figure 5: Should be a 1-column figure Figure 6: Should be a 1-column figure Figure 7: Should be a 1-column figure Figure 8: Should be a 1-column figure Figure 9: Should be a 1-column figure The four Graphics following Figure 9: Should all be a 1-column figure Figure 10: Should be a 1-column figure Figure 11: Should be a 1-column figure Figure 12: Should be a 1-column figure Figure 13: Should be a 1-column figure Figure 14: Should be a 1-column figure The two graphics following Figure 14: Should be a 1-column figure Equation (1): Should be a 1-column figure Figure 15: Should be a 1-column figure Figure 16: Should be a 1-column figure Figure 17: Should be a 2-column figure Figure 18: Should be a 2-column figure Graphic after Figure 18: Should be a 1-column figure Figure 19: Should be a 1-column figure Figure 20: Should be a 1-column figure Figure 21: Should be a 2-column figure Figure 22A: Should be a 1-column figure Figure 22B: Should be a 1-column figure


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51 274. Parkin, A., Goldet, G., Cavazza, C., Fontecilla-Camps, J. C., and Armstrong, F. A. (2008) The difference a Se makes? Oxygen-tolerant hydrogen production by the [NiFeSe]hydrogenase from Desulfomicrobium baculatum, J. Am. Chem. Soc. 130, 13410-13416. 275. Marques, M. C., Coelho, R., De Lacey, A. L., Pereira, I. A., and Matias, P. M. (2010) The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, “as-isolated”state, J. Mol. Biol. 396, 893−907. 276. Sivaramakrishnan, S., Ouellet, H., Du, J., McLean, K. J., Medzihradszky, K. F., Dawson, J. H., Munro, A. W., and Ortiz de Montellano, P. R. (2011) A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid, Biochemistry 50, 3014−3024. 277. Eckenroth, B. E., Rould, M. A., Hondal, R. J., and Everse, S. J. (2007) Structural and biochemical studies reveal differences in the catalytic mechanisms of mammalian and Drosophila melanogaster thioredoxin reductases, Biochemistry 46, 4694-4705. 278. Eckenroth, B. E., Harris, K., Turanov, A. A., Gladyshev, V. N., Raines, R. T., and Hondal, R. J. (2006) Semisynthesis and characterization of mammalian thioredoxin reductase, Biochemistry 45, 5158-5170. 279. Berner, R. A., Vandenbrooks, J. M., and Ward, P. D. (2007) Evolution. Oxygen and evolution. Science 316, 557-558.

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