Why Nature Chose Selenium - UW-Madison Chemistry · Why Nature Chose Selenium Hans J. Reich*, ‡...

Why Nature Chose SeleniumHans J. Reich*, ‡ and Robert J. Hondal*,†

†University of Vermont, Department of Biochemistry, 89 Beaumont Ave, Given Laboratory, Room B413, Burlington, Vermont 05405,United States‡University of WisconsinMadison, Department of Chemistry, 1101 University Avenue, Madison, Wisconsin 53706, United States

ABSTRACT: The authors were asked by the Editors of ACSChemical Biology to write an article titled “Why Nature ChoseSelenium” for the occasion of the upcoming bicentennial ofthe discovery of selenium by the Swedish chemist Jons JacobBerzelius in 1817 and styled after the famous work of FrankWestheimer on the biological chemistry of phosphate[Westheimer, F. H. (1987) Why Nature Chose Phosphates,Science 235, 1173−1178]. This work gives a history of theimportant discoveries of the biological processes that seleniumparticipates in, and a point-by-point comparison of thechemistry of selenium with the atom it replaces in biology, sulfur. This analysis shows that redox chemistry is the largestchemical difference between the two chalcogens. This difference is very large for both one-electron and two-electron redoxreactions. Much of this difference is due to the inability of selenium to form π bonds of all types. The outer valence electrons ofselenium are also more loosely held than those of sulfur. As a result, selenium is a better nucleophile and will react with reactiveoxygen species faster than sulfur, but the resulting lack of π-bond character in the Se−O bond means that the Se-oxide can bemuch more readily reduced in comparison to S-oxides. The combination of these properties means that replacement of sulfurwith selenium in nature results in a selenium-containing biomolecule that resists permanent oxidation. Multiple examples of thisgain of function behavior from the literature are discussed.

■ PREFACEThe authors were asked by the Editors of ACS Chemical Biologyto write an article titled “Why Nature Chose Selenium,” styledafter the famous work of Frank Westheimer titled “Why NatureChose Phosphates.”1 While Westheimer’s elegant chemicalexplanations for the use of phosphate in biology have foundbroad acceptance, currently the chemical reasons for the use ofselenium in biology remain elusive and not widely agreedupon.2−16 This work is written for the occasion of theupcoming bicentennial of the discovery of selenium by theSwedish chemist Jons Jacob Berzelius in 1817. We hope readersof this review on the chemistry of the “mysterious moonmetal”17 will be illuminated by our views.

■ DISCOVERY OF SELENIUMOldfield describes the discovery of selenium by Berzelius as“Serendipity,” because he claims it was discovered during aninvestigation into an illness of the workers in a chemical factoryat Gripsholm, Sweden (in part owned by Berzelius) thatproduced acetic, nitric, and sulfuric acids. As related byOldfield, this illness was precipitated when the factory switchedto a new, local source of sulfur ore.18 As the story goes,Berzelius thought this illness might be due to arseniccontamination of this sulfur ore, and the analysis of this oreled to the isolation of a new element (selenium). This storymay be apocryphal, as it is not mentioned by Trofast, who hasreported on the discovery of selenium from a careful study ofBerzelius’ original notes.19 Trofast reports that Berzelius

declared, “...I, to mark its akin properties with tellurium, havenamed selenium, from Σεληνη, moon (goddess). What is more,it is in this regard, midway between sulfur and tellurium, andhas 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 ofcompounds. This theory held that all chemical compoundswere held together due to neutralization of opposite electricalcharges, as does occur in ionic compounds. It is tempting tothink that the naming of selenium was a type of homage to thistheory as tellurium had been named after Tellus, the Latingoddess of the Earth (Earth Mother). Ultimately, electro-chemical dualism could not describe all types of chemicalbonding and fell out of favor as a theory, but Berzelius’discovery of selenium remains as a significant achievement inchemistry.

■ EARLY STUDIES OF SELENIUM IN BIOLOGYThe first recognized role of selenium in biology was as a toxin.The investigation into the cause of “alkali disease” and “blindstaggers,” diseases of livestock in the American West and PlainsStates by Kurt Franke and others, showed that these diseaseswere forms of selenosis due to the ingestion of high doses ofselenium found in cereal crops, animal forage, and selenium

Received: January 12, 2016Accepted: March 7, 2016Published: March 7, 2016

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© 2016 American Chemical Society 821 DOI: 10.1021/acschembio.6b00031ACS Chem. Biol. 2016, 11, 821−841

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accumulator plants such as Astragalus (known commonly as“locoweed”) grown in soils with high selenium content.21−24 Itis remarkable that Franke at a very early date was able to showthat the toxic form of selenium in locally grown grains was inthe protein fraction of sulfuric acid hydrosylates. His experi-ments showed that selenium was “adsorbed on the proteinmolecule.”25 He was able to conclude that “There is evidencethat most of the selenium is in a compound very similar tocystine.”26 Franke’s prescience that selenium would replace thesulfur atom of an amino acid is little recognized26 and predatesthe discovery of the “21st” amino acid,27,28 selenocysteine, byThressa Stadtman29 by 40 years! It should be noted that, whileblind staggers is often attributed to selenosis, it may in fact becaused by sulfate-related polioencephalomalacia due tocontamination of water sources by sodium sulfate andmagnesium sulfate.30

■ SELENIUMTOXIC AND ESSENTIAL

Selenium, like the moon, has two faces,31 as it is both toxic toall organisms and essential to many bacteria and animal species.The essentiality of selenium to bacteria was to be discovered byPinsent, who found that selenium was necessary for the activityof E. coli formate dehydrogenase in 1954.32 A few years later,selenium was discovered to be essential to animalsindependently by Patterson33 and Schwarz.34,35 Karl Schwarz,who even earlier was studying dietary liver necrosis in rats, hadfound that the addition of methionine, vitamin E, or a “thirdfactor” to the diet could prevent this condition.36 It is theidentification of this “third factor” that Schwarz would becomeremembered for. Schwarz moved from Germany to the UnitedStates and took a position at the National Institutes of Healthinvestigating the cause of exudative diathesis in chicks and livernecrosis in rats, diseases that were precipitated by a diet oftorula yeast. Torula yeast is low in vitamin E, selenium, andsulfur amino acids, but rich in unsaturated fatty acids.37 Thesediseases did not occur if American brewer’s yeast (S. cerevisiae)was used instead. Schwarz was working on identifying themissing factor found in brewer’s yeast that prevented thesediseases.38 Schwarz initially thought that this missing factormight be a vitamin, but experiments showed that the missingsubstance must be an inorganic compound. One of threeelements, arsenic, selenium, and tellurium were suspected asthe missing nutritional factor.38 Schwarz isolated the missingfactor from acid hydrolysates of protein and called it “Factor 3”because it was the third substance identified that could preventdietary liver necrosis.35 Jukes relates the story that Schwarz wasable to identify selenium as “Factor 3,” the nutrient needed toprevent liver necrosis in rats, after Dr. DeWitt Stetten, then anAssociate Director of the National Institute of General MedicalSciences, walked into Schwarz’s laboratory and smelled thedistinct odor of a selenium-containing compound emanatingfrom open test tubes of “Factor 3” in his laboratory.38 The odormay have been from dimethyl diselenide, which has a verysharp odor and is a decomposition product of selenomethio-nine.The two faces of selenium, essential and toxic, are unique in

that the range between the amounts needed to maintain healthor cause toxicity is quite narrow. The U.S. Department ofAgriculture has a R.D.A. of 55 μg/day for adults,39 while theWorld Health Organization has established a toxic limit of 800μg/day for adults.40 For this reason, Jukes refers to selenium asthe “essential poison.”41

■ DISEASES OF SELENIUM DEFICIENCY

Besides exudative diathesis and liver necrosis, seleniumdeficiency results in a number of other diseases of animalsand humans.42 These include white muscle disease (a musculardystrophy disease mainly of sheep); mulberry heart disease, adisease affecting animal livestock and is so named due tohemorrhage of the heart that gives the organ the color andappearance of a mulberry;42 and in humans, KeshanDisease43−46 and Kashin−Beck Disease.47 Keshan Disease is atype of cardiomyopathy and may have an underlying viraletiology that is associated with selenium deficiency.48,49

Kashin−Beck Disease is an osteoarticular disorder thatresembles rheumatoid arthritis in some respects but is muchmore severe. The beginning stages of the disease may involvedestruction of the cartilage of the joints. The exact underlyingcause of the disease is not known with certainty, but the diseaseis strongly associated with both selenium and iodinedeficiency.50 It was the discovery of Keshan Disease andmammalian selenium-containing proteins that establishedselenium as an essential trace element for humans.45,46,51−53

■ CONNECTION WITH VITAMIN E

Although it has been shown independently by McCoy andThompson that there is a biochemical function of selenium thatmust be distinct from that of vitamin E,37,54 the presence ofvitamin E can prevent, or attenuate, various animal diseases thatare associated with selenium deficiency.55−68 This implies thatat least one biochemical function of selenium is stronglyconnected with that of vitamin E. With the discovery ofglutathione peroxidase as a selenoenzyme,52,53 it became clearthat one common function of the two is protection against lipidperoxidation.68 Another biochemical connection betweenselenium and vitamin E is vitamin C (ascorbic acid). Thereduction of dehydroascorbic acid to ascorbic acid is catalyzedby thioredoxin reductase, a selenoenzyme.69 Ascorbic acid inturn can reduce the vitamin E radical formed in lipid bilayersafter quenching a radical species. It is interesting to note thatthe coxsackievirus implicated as the underlying cause of KeshanDisease mutates to a more virulent form when the host isdeficient in either selenium or vitamin E.70 This result couldmean that both nutrients protect the host DNA from amutation associated with an oxidation event, which leads to amore virulent form of the virus. Alternatively, Loscalzo hasoffered a possible mechanism that does not involve mutation ofthe virus. Low levels of glutathione peroxidase expression dueto selenium deficiency can result in oxidative stress that leads tomyocardial injury and ventricular dysfunction, which in turnleads to cardiomyopathy characteristic of Keshan Disease.71

■ SELENIUM−CANCER HYPOTHESIS

There has been a great deal of interest in the area of cancerchemoprevention by selenium since the late 1960s. The earliestreport of a relationship between selenium and cancer was byNelson and co-workers,72 who reported that a high dietaryintake of selenium caused liver tumors in rats. However,experiments conducted later in the same decade also on ratsshowed that low doses of sodium selenite (Na2SeO3) protectedagainst tumors induced by injection with dimethylaminoazo-benzene (a 50% reduction in tumor incidence was reported).73

Then in 1966, Shamberger and Rudolph showed that sodiumselenide (Na2Se2) applied in a topical solution greatly reduced

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(730-fold) tumor formation in an induced mouse skin tumormodel compared to DL-α-tocopherol.74

To help resolve the question of whether or not low levels ofselenium were carcinogenic or not, the National CancerInstitute funded several studies that showed that selenium inthe diet up to 8 ppm did not induce tumorigenesis,75−77

although there was still fear about the subject of seleniumtoxicity among the general public and some in the scientificcommunity even after these studies.78 In a very influential letterto the editor of the Canadian Medical Association Journal,Shamberger and Frost hypothesized that, “If selenium had aneffect on public health, areas adequate or deficient could beexpected to show different disease incidences or death rates.”79

They pointed to a then recent study by Kubota and co-workerswho had constructed a forage crop map of the U.S. thatindicated which areas of the country had high or lowselenium.80 Using these data, they showed a correlationbetween areas of the U.S. with low forage crop selenium anda higher death rate. They also highlighted a study by Allawayand co-workers who had measured plasma selenium levels inmultiple cities and counties in the U.S., and these measured lowplasma selenium levels were correlated with higher cancer deathrates.81 These ideas prompted Schrauzer to do a global studythat examined the relationship between dietary selenium intake(they also measured plasma selenium) and cancer. He foundinverse correlations for cancers of the large intestine, rectum,prostate, breast, ovary, and the lung.82 With respect todeficiency of selenium in soils, it should be noted that this issuch a concern in Finland that the government has mandatedthe inclusion of selenium in fertilizer for agricultural land.83 Tocombat Keshan Disease in low selenium areas of China,Chinese health officials began adding sodium selenite to tablesalt.45

In the decades since 1970, numerous epidemiological,selenium supplementation studies, and clinical trials mostlysupported the link between low selenium intake and a higherincidence of cancer (termed the selenium−cancer hypothesis).There are far too many examples of these kinds of studies togive a complete listing here, but some important ones are givenin the reference list.84−94 Willett and Stampfer, Clark andAlberts, Jackson and Combs, Schrauzer, and Ip give goodsummaries of the issues surrounding selenium and chemo-prevention as well as a review of some of the important workdone in this area.95−99

The selenium-cancer hypothesis perhaps reached its zenith in1996 with a study led by Clark and Combs that showed thatsupplementation with 200 μg/day of selenium in the form ofselenized yeast led to significant reductions in colon, prostate,and lung cancers in a multicenter, double-blind, randomized,placebo-controlled cancer prevention trial.100 Notably, aKaplan−Meier curve showed that selenium supplementationresulted in significant reductions in total cancer mortality (i.e.,increased survival probability) over a 10 year time period.100 Inresponse to this very positive outcome, the National Institutesof Health undertook an extremely large (35 533 men)randomized, placebo-controlled selenium supplementationtrial. This trial was named the Selenium and Vitamin E CancerPrevention Trial (SELECT).101 An important distinctionbetween the earlier trial and the SELECT study was the useof 200 μg/day of L-selenomethionine as the source of seleniuminstead of selenized yeast. The study found that there were nosignificant differences in any of the cancer end points. In otherwords, they did not find evidence that supplementation with

selenium offered any protection against cancer.102 This resultwas extremely disappointing (to say the least) and contrary tomany previous studies that supported the selenium−cancerhypothesis.Hatfield and Gladyshev have discussed some of the reasons

for the large difference in experimental outcomes between theprevious work (especially the work by Clark and co-workers)and the SELECT study.103 They note three significantdifferences: (i) The study by Clark et al.100 was initiallyundertaken to examine the effect of selenium supplementationfor those at risk for skin cancer and so only considered riskfactors for skin cancer during the randomization of subjects. (ii)The SELECT study used a different form of selenium,selenomethionine, while the study by Clark et al. used selenizedyeast. While selenomethionine can be used to make selenium-containing proteins, other forms of selenium could beimportant for chemoprevention of cancer. (iii) Last, partic-ipants in the SELECT trial had higher initial plasma levels ofselenium than those in the study by Clark et al. This last factsuggests that supranutritional dietary selenium does not providecancer protection, though epidemiology indicates that seleniumdeficiency can increase cancer incidence (vide supra).One seemingly contradictory fact about selenium and cancer

is that overexpression of multiple selenoproteins such asglutathione peroxidase-2, Sep15, and thioredoxin reductase mayhelp to promote cancer growth once the tumor has takenhold.103−105 The fact that increased expression of aselenoenzyme such as thioredoxin reductase might help supporttumor growth highlights an interesting fact about cancer cellsand selenium. Cancer cells produce more reactive oxygenspecies (ROS) than normal cells and are adapted to a higherlevel of endogenously produced oxidants.106,107 Thioredoxinand thioredoxin reductase are overexpressed in many humancancer types,108,109 and this important selenium-containingantioxidant system helps to counteract oxidative stressexperienced by cancer cells and enables cancer cells to resistprogrammed cell death (apoptosis). This fact contradicts theoriginal idea of why selenium might help to prevent cancer;selenium, as part of antioxidant enzymes, helps to preventoxidative damage to DNA by free radicals and ROS. However,selenium can be involved in killing cancer cells using theopposite mechanism. Selenolates can react with molecularoxygen to produce superoxide,110 and the superoxide may pushthe cancer cell over an “oxidative cliff” from which the cellcannot recover, causing it to undergo apoptosis.107,111 Indeed,there are clinical trials currently being undertaken to treatcancer that take advantage of this chemical reaction withselenium using sodium selenite.112,113 Selenite and methanese-leninic acid, common forms of selenium in biology, are alsovery good oxidants. Both can oxidize thiol groups of enzymes,which could help push cancer cells toward apoptosis. Here, wesee two more “faces” of selenium, as an antioxidant and anoxidant.

■ FORMS OF SELENIUM IN BIOLOGYA very nice short review of the subjects discussed above can befound in ref 114. We now turn to the chemical forms ofselenium used in biology and the types of chemistry seleniumcan perform. There are multiple chemical forms of seleniumused in biology. Eight of these forms are shown in Figure 1.The principal form is that of selenocysteine, the 21st aminoacid in the genetic code where it is cotranslationally insertedinto the polypeptide chains of selenoproteins.27,28 In addition



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to being incorporated into proteins, selenium is found innucleic acids, specifically as 5-methylaminomethyl-2-selenour-idine (mnm5Se2U), where it is found in the wobble position ofthe anticodon loop in tRNAGlu, tRNALys, and tRNAGln innumerous species of bacteria.115−119 The selenolate ofselenocysteine is a ligand for a number of coenzymes inbacteria such as in (i) the molybdenum atom of molybdopteringuanine dinucleotide in formate dehydrogenase,120−124 (ii) thenickel atom of NiFeSe hydrogenases,125,126 and (iii) iron in aputative iron−sulfur cluster in the methionine sulfoxidereductase from Metridium senile.127 Selenium is also found asthe analog of ergothioneine in tuna, named selenoneine.128

This novel selenium biomolecule may be involved in mercurydetoxification in fish.129 A methylated form is present inhumans, but its function is not known.130 A very importantdietary source of selenium is selenomethionine. Plants convertinorganic forms of selenium into selenomethionine, which isthen converted into selenocysteine in animals via the trans-sulfuration pathway (reviewed in ref 131). Selenocysteineproduced by this pathway is then converted into hydrogenselenide, which combines with ATP to produce selenophos-phate132−134 in a reaction catalyzed by selenophosphatesynthetase.135−137 In bacteria (e.g., E. coli), selenophosphateis used as the nucleophile to attack the carbon−carbon doublebond of dehydroalanyl-tRNA[Ser]Sec, yielding selenocysteyl-tRNA[Ser]Sec.138 This specialized tRNA brings selenocysteineto the ribosome where it is incorporated into selenocysteine-containing proteins. Selenocysteyl-tRNA[Ser]Sec is also usedto synthesize selenoproteins in eukaryotes, but a dehydroala-nine-containing tRNA is not used as the acceptor for the attackby selenophosphate. Instead, a phosphate group on O-phosphoseryl-tRNA[Ser]Sec is displaced by selenophosphateto produce selenocysteyl-tRNA[Ser]Sec.139,140 Alliums such asgarlic and onions tend to concentrate inorganic selenium in Se-methylselenocysteine141 (not shown), which is converted intoselenophosphate by a pathway that utilizes selenocysteine β-lyase and methaneselenol demethylase.142

Other forms of selenium in biology not shown in Figure 1are (i) selenocysteine as a ligand for the related molybdopter-

in−cytosine dinucleotide in carbon monoxide hydroge-nase,143,144 (ii) Se-methyl-N-acetylselenohexosamine, a seleno-sugar that is the major excretory selenium metabolite found inurine,145,146 (iii) excretory compounds dimethyl selenide(breath) and trimethylselenonium (urine),147,148 (iv) selenite,which can react with glutathione to produce selenodigluta-thione,149 and (vi) Se-methylselenocysteine, which in animalscan be converted to methaneselenol by selenocysteineconjugate β-lyases.150,151 A more complete list of biologicallyimportant selenocompounds can be found in ref 131. Seleniumfrom methaneselenol can be converted into excretory formsdimethyl selenide and trimethyselenonium, or it can beconverted into selenophosphate and put into selenoproteins.152

Ip and Ganther have compiled a considerable amount of dataimplicating methaneselenol as the form of selenium that isanticarcinogenic.153−156 This may be due to redox cycling ofthis compound that induces apoptosis due to the formation ofsuperoxide.157

■ WHY SELENIUM? CLUES FROM THESELENOCYSTEINE INSERTION MACHINERY ANDBIOGEOCHEMISTRY

Selenocysteine, a major form of biological selenium, is a trueproteinogenic amino acid because it meets the criteria met bythe other 20 common amino acids: (i) it is encoded by DNAand it has its own unique codon (UGA); (ii) it has a uniquetRNA that brings the aminoacylated selenocysteine residue tothe ribosome; (iii) is cotranslationally inserted into thepolypeptide chain at the ribosome.158 The insertion ofselenocysteine is much more complicated than cysteine, andthe other 19 proteinogenic amino acids as shown in Figure 2.

Figure 1. Different chemical forms of selenium used in biomolecules.(1) Selenocysteine (Sec, U). (2) 5-Methylaminomethyl-2-selenour-idine. (3) Selenium, as selenocysteine, is a ligand for themolybdopterin guanine dinucleotide cofactor of formate dehydrogen-ase. (4) Selenium, as selenocysteine, is a ligand for nickel in [NiFeSe]hydrogenases. (5) Selenium, as selenocysteine, is a putative ligand foriron in an iron−sulfur cluster. (6) Selenium is found in selenoneine,the selenium analog of ergothioneine. (7) Selenomethionine (SeMet).(8) Monoselenophosphate.

Figure 2. Eukaryotic Sec-insertion machinery. In eukaryotes,phosphoseryl-tRNASec kinase (PSTK) phosphorylates aminoacylatedserine to form O-phosphoseryl-tRNA. Sep (O-phosphoserine)tRNA:Sec (selenocysteine) tRNA synthase, abbreviated as SepSecS,then converts O-phosphoseryl-tRNA to Sec-tRNA, using selenophos-phate 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 ofa special stem-loop structure in the 3′-untranslated region of themRNA (SECIS element) and a SECIS binding protein (SBP2). Thedetails of Sec-insertion were first characterized in bacteria,161−165 andit should be noted that there are substantial differences in the Sec-insertion machineries of prokaryotes and eukaryotes.166−173 We alsonote that eukaryotes require additional accessory proteins for Sec-insertion not depicted here.172,173



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The codon for selenocysteine is UGA, normally a stopcodon. This UGA stop codon must be recoded as a sensecodon for selenocysteine, and this recoding process requires anelaborate apparatus involving numerous accessory proteins anda special signal in the 3′-untranslated region of the mRNA ofthe selenoprotein.159−169 The details of the selenocysteine-insertion machinery are reviewed in refs 170−173. Second, it isextremely costly in terms of the cellular energy currency, ATP,to insert selenocysteine into a protein. It costs ∼25 mol of ATPto insert 1 mol of cysteine into a protein.174 Given the multipleaccessory proteins required to insert selenocysteine into aprotein, the biosynthetic costs of a selenoprotein must beconsiderably more than that of a cysteine-containing protein. Athird consideration for biology is the geological distribution ofselenium in the Earth’s crust, as sulfur is much more abundantrelative to selenium. This ratio is estimated to be as low as6000:1175 and as high as 55 500:1.176 In addition, selenium isnot distributed evenly over the Earth’s crust.177 For example,there are both seleniferous and selenium deficient areas ofChina and the American west. Selenium deficient soils areespecially consequential in China, New Zealand, and Finland.83

This means that animal life on land does not have equal accessto this essential nutrient.Considering the three factors mentioned above, it is natural

to ask the question, “why did nature choose selenium?” Theanswer of the authors is that selenium must be able to performsome chemical function necessary for biology that sulfur is notvery good at. In other words, there is a very large chemicaldifference between the two elements. If the chemical differencesbetween selenium and sulfur were small, then nature couldabandon the use of selenium and not be dependent upon thefactors listed above and use sulfur instead. The catalytic activityof the sulfur-containing enzyme may (or may not!) be lowerthan that of the selenium-containing ortholog, but nature couldcompensate by making more of the sulfur version of theenzyme if needed. In the following sections, we review thechemical differences between sulfur and selenium.

■ SELENIUM-CONTAINING ENZYMESWhile selenium is found in a variety of biomolecules as notedabove, many of its important biological functions are due to itsuse in proteins, and this is where our discussion will be focused.Most selenium-containing enzymes make use of the nucleo-philic and reducing properties of the selenolate (Sec-Se−) formof a selenocysteine to perform redox reactions. After beingoxidized, the resulting selenenic acid (Sec-SeOH) oxidationstate is typically returned to selenolate by reduction withglutathione or a resolving Cys residue on the enzyme. The beststudied selenoenzymes are the glutathione peroxidases (Gpx),iodothyronine deiodinases (DIO), thioredoxin reductases(TrxR), and methionine sulfoxide reductases (Msr). Thereare eight Gpx isozymes in humans, five of which containselenium,178 and they are an essential part of the system thatscavenges hydroperoxides and hydrogen peroxide to preventoxidative damage. There are three human Sec-containingiodothyronine deiodinases179 found in the thyroid gland andother tissues, and they function to reduce the aryl iodide bondsof thyroxine (T4) and triiodothyronine (T3) to a C−H. Thereare three human Sec-containing thioredoxin reductases: acystolic form, a mitochondrial form, and a specialized testes-specific enzyme.180 TrxR’s help to maintain thiol−disulfideredox homeostasis via reduction of the small proteinthioredoxin (Trx). Depending on the form of the enzyme,

Msr reduces either free methionine sulfoxide or peptidylmethionine sulfoxide to methionine. There are four humanMsr’s, only one of which contains Sec.181 The Sec-containingMsr (MsrB1) is stereospecific for methionine-R-sulfoxide. Thereduction of methionine-R-sulfoxide on actin promotes actinpolymerization.182 Figure 3 shows the chemical processes

shown to be, or likely to be, involved in the Sec-catalyzedreactions. 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 involvesnucleophilic 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 actsas an electrophile, being attacked by a thiolate, and the catalyticcycle is completed by a reaction where selenolate behaves as aleaving group in the final reduction step of the catalytic cycle. Itcan be shown that selenium is likely to be more effective thanthe sulfur analog as a nucleophile, or as a leaving group, but notdramatically so. Typical Se/S rate ratios are 1 or 2 orders ofmagnitude (vide inf ra). It should be pointed out that whileselenocysteine is found in the three major divisions of life,archeabacteria, eubacteria, and eukaryotes, there are entireclasses of organisms that lack selenocysteine (Lepidoptera forexample) where transformations such as the above areperformed by cysteine.183,184

■ CHEMICAL PROPERTY COMPARISONS BETWEENSULFUR AND SELENIUM

Even long before the interesting chemical and biologicalquestion “why selenium?” was raised, comparison of theproperties of sulfur compounds and their selenium analogs haddrawn the attention of many chemists interested in selenium,

Figure 3. Chemical processes mediated by Sec-containing enzymes(Enz-Se−, selenocysteine moiety of enzyme; GS−, glutathione; Trx,thioredoxin: B+−H, a proton donor).



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often with the intent of identifying chemical properties thatmight make the selenium compounds useful. In many respectssulfur and selenium have very similar physical and chemicalproperties:10 they share all of the same oxidation states andfunctional group types (Figure 4), and their structures are oftenso similar that analogous compounds can easily cocrystallize.Here, we are interested in the significant chemical differencesbetween the two chalcogens that might justify the metaboliccost of utilizing selenium. We will summarize some of thosethat have relevance to reactions of biological interest.Many of the significant differences between selenium and

sulfur are a consequence of the usual changes on going fromlighter to heavier elements. Heavier elements are morepolarizable (“softer”) than lighter ones, and this usually leadsto more rapid electrophilic and nucleophilic substitutions at theelement. Most bond strengths to selenium are weaker thanthose to sulfur, and this results in substantially faster bond-breaking reactions. The weaker bonds to selenium mean thatthe sigma* orbital of the Se−X bond is lower in energy thanthat of a S−X bond, hence more reactive as an electronacceptor. Thus, all oxidation states of selenium are much moreelectrophilic compared to sulfur analogs. It is also generallyobserved that higher oxidation states become relatively lessstable for the heavier elements, and this is also true for seleniumvs sulfur. Heavier elements are also more tolerant ofhypervalent bonding situations.The single most important use of selenium by synthetic

organic chemists, the selenoxide elimination (Figure 5) to formalkenes, occurs at ca. 100 °C milder temperatures and is ca.100 000 times as fast as related sulfoxide eliminations.185,186

■ ACIDITY

The weaker bond to hydrogen, together with the increase insize and polarizability of the heavier atom, results insubstantially 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 fullyionized to a selenolate.

■ NUCLEOPHILICITYIn spite of their lower basicity, selenolate ions are morenucleophilic by roughly 1 order of magnitude than thiolates,presumably a consequence of the higher polarizability ofselenium (Figure 7). This is true for SN2 substitutions189,190 aswell as for aromatic substitutions.191 In protic solvents theweaker hydrogen bond acceptor properties of selenolates vsthiolates contribute to higher nucleophilicity. Selenides are alsomore nucleophilic than sulfides.189

Figure 4. Structures and oxidation numbers of sulfur and selenium compounds that result from two-electron oxidation reactions. Arrows show theconversion pathways between oxidation states. The open circle represents carbon, and the number inside the circle represents the oxidation numberof sulfur or selenium that it is attached to. The magenta X denotes a slow reaction. The [O] symbol represents a two-electron oxidant.

Figure 5. Relative rates of selenoxide185 and sulfoxide186 syn-eliminations. Y = S, or Se, here, and throughout the text.

Figure 6. Comparison of selenol and thiol pKa values.

Figure 7. Comparison of selenium and sulfur nucleophilicities in SN2substitutions using phenyl selenolate/thiolate,189 thioselenolate/thiocyanate,190 dimethyl selenide/sulfide with methyl iodide,189 andphenyl selenolate/thiolate with 4-nitro-1-bromofuran.191 Selenocys-teine is also more nucleophilic then cysteine in a substitution at an acylcarbon.192



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There is a much more significant difference in nucleophilicitybetween the two chalcogens at physiological pH, as selenols arecompletely converted to selenolates, whereas thiols are onlyslightly ionized. Since the selenolates/thiolates are almostcertainly the active nucleophile, selenolates have the doubleadvantage of both higher intrinsic nucleophilicity and a muchhigher fraction of the active form. In direct comparisons, thisleads to ∼2 orders of magnitude higher reactivity for selenolatesversus thiols around pH 7. Some examples of this increasedreactivity using selenocystamine (9) and cystamine (10) areshown in Figure 8. While nature may take advantage of this

difference in nucleophilicity at physiological pH, the pKa valuesof the nucleophilic cysteines in the context of a proteinmicroenvironment can be greatly perturbed as evidenced by thepKa values of the active site Cys residues of papain, caricain, andficin, which are 3.3, 2.9 and 2.5, respectively.193 As such, naturemay be able to increase the nucleophilicity of sulfur so as tominimize this difference at physiological pH,194 and it wasconcluded that selenium is not a “chemical necessity” inTrxR.195

■ LEAVING GROUP ABILITYSince selenolate is less basic than thiolate, selenolates areusually better leaving groups. As shown in Figure 9a, the selenolester 11 (Y = Se) decomposes to ketene 180-fold faster thanthe thiol ester 11-S,197 while in the biologically more relevant

example in Figure 9b ,the selenosulfide (also referred to as aselenenylsulfide) is only 3-fold more reactive than thedisulfide.196 This relatively small number may in part be dueto stronger hydrogen bonding of the more basic thiolate at thetransition state.

■ HYPERVALENCYOne of the few bonding situations where Se−X bonds arestronger than S−X bonds occurs in hypervalent compounds,i.e., where the number of bonds plus lone pairs is greater thanthat allowed by the Lewis octet rule. Thus, selenuranes (R4Se)form more easily than sulfuranes (R4S) and are much morestable. 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 factorsare smaller steric effects due to the longer bonds to seleniumand a lower LUMO sigma* orbital which leads to a morefavorable three-center four-electron hypervalent bondingsituation. This greater tolerance for hypervalency (also sharedby the neighboring element pairs Cl/Br and P/As, and whichcontinues with the heavier elements Te, I, and Sb) can be seenin many contexts. For example, the hypervalent sulfurane 12-Sdecomposes at −67 °C with a half-life of 46 min, while theselenurane 12-Se has to be “heated” to 0 °C before thedecomposition rate is comparable, corresponding to a differ-ence in activation energy of 2.9 kcal/mol.199 The additionproduct of chlorine and dialkyl sulfides (e.g., the Corey-Kimreagent200) is an ionic structure 13, whereas that of dialkylselenides is covalent (14).201 Another occurrence of strongerSe−X bonds occurs in bonding to metals. For example, Hg−Sebonds are stronger than Hg−S bonds.202

■ ELECTROPHILICITYThe greater tolerance for hypervalency of selenium has animportant consequence, that nucleophilic attack on selenium(which typically forms or passes through hypervalentintermediates such as R4Se or R3Se

−) usually occurs muchmore rapidly than at sulfur, since the intermediate seleniumcompounds are lower in energy than sulfur analogs.203 Thus,selenium compounds of all types are much better electrophilesthan sulfur analogs, and this can be seen in various contexts.For example, bis(phenylthio)methane 15 is deprotonated withn-BuLi,204 whereas the selenium analog 16 is attacked atselenium.205

In a closely related comparison, the rate of Ph/Tolylexchange of diphenyl selenide (17-Se) with tolyllithium is >2 ×104 times as fast as with diphenyl sulfide (17-S).206

Figure 8. Comparison of nucleophilic substitution by selenolate/thiolate on a disulfide and a peroxide.196 These reactions were donenear neutral pH, where the cystamine (10) is largely in the SH form.When corrected to pH 10,15 the relative rates are much smaller.

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.



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Nucleophilic substitutions at a selenenyl halide such as 18-Seis substantially faster than at the analogous sulfenyl center, withkSe/kS ranging from 6000 for a dithiocarbamate to 150 forcyanide.207 Similarly, nucleophilic attack of cyanide onPhSeSO2Ar is 5 orders of magnitude faster than onPhSSO2Ar.

208

■ COMBINATION OF NUCLEOPHILICITY,ELECTROPHILICITY, AND LEAVING GROUPABILITY

In a very careful study of the pH-dependence of the degenerateexchange of cystamine (10) with cysteamine and selenocyst-amine (9) with selenocysteamine, all three effects are operatingin the same direction in the case of the selenolate/diselenideexchange reaction: the higher nucleophilicity of selenolate atphysiological pH, the better leaving group ability of selenolate,and the stronger intrinsic electrophilicity of the center seleniumatom. Using dynamic NMR techniques (line broadening forselenium, saturation transfer for sulfur), the selenolate/diselenide exchange reaction was measured to be 107 times asfast as that of the thiol/disulfide exchange reaction at pH 7.209

■ WEAKER π-BONDINGThe larger size of selenium compared to sulfur (atomic radiusof 115 pm compared to 100 pm for sulfur) results in largerhybridized orbitals, and this plus the longer bond length leadsto weaker π overlap. One consequence of this is thatselenoesters are much less stabilized than thioesters byresonance with the carbonyl group.210 As shown in Figure10, the acyl transfer between selenocystamine (9) and

cystamine (10) strongly favors the thioester. Both nature andchemists have taken advantage of the high reactivity ofselenoesters as acyl transfer reagents. Selenoesters have beenused to catalyze native chemical ligation reactions,211−213 andselenoprotein K uses its Sec residue to form a selenoester tocatalyze acyl-transfer of a palmitoyl group to a calciumchannel.214

■ REDOX PROPERTIESThe greatest divergence between selenium and sulfur chemistryoccurs in the redox reactions of the two elements. This is truefor both two-electron and one-electron processes. We willbegin our discussion with two-electron oxidation reactions.

One of the consequences of the longer bond length ofheavier elements is a reduced ability to form π-bonds of alltypes as introduced above. This means that even though achalcogen-oxygen double bond is commonly depicted forconvenience (as in Figure 4), a much better representation ofthe electronic structure of these compounds, particularly forselenium, is given in Figure 11.

Presumably because of stronger back-donation of the lone-pair electrons on oxygen to acceptor orbitals (sigma* andpossibly d orbitals) on sulfur compared to selenium, the Y−Odative bonds in selenoxides, selenones, seleninic acids, andselenonic acids are weaker for selenium, have substantially moredipolar character, and are relatively less favored than for sulfur.This can be seen in many property changes when S/Secomparisons are made. For example, alkyl selenones areexcellent alkylating agents,215,216 while sulfones are completelyunreactive.The differences in electronic structure noted above in the

chalcogen oxides leads to large differences in their chemicalproperties. For example, dimethyl selenoxide (pKa of conjugateacid 2.55217) is substantially more basic than dimethyl sulfoxide(pKa of conjugate acid −1.54218). Thus, in an acid-catalyzedreaction at a given pH, selenoxides will have ca. 104 as high aconcentration of the reactive protonated selenoxide comparedto the protonated sulfoxide. This would already result indramatically higher reactivity, but in addition the seleniumbears more positive charge and is inherently more electrophilicthan sulfur, combining to give very much higher rates ofnucleophilic attack at selenium.Another example of the enhanced reactivity of the Se-oxide

relative to an S-oxide is the racemization of selenoxidescompared to sulfoxides. The acid-catalyzed racemization ofsulfoxides is a slow and difficult process. Selenoxides racemizemany orders of magnitude faster than the correspondingsulfoxides. The data for the selenoxide inversion in Figure 12were obtained from dynamic NMR measurements, whereas thesulfoxide data were obtained from the epimerization of onediastereomer to the other. The inversions proceed by differentmechanisms; the selenoxide is first order in [H+] independentof whether HCl or H2SO4 was used, so it follows path A. Thesulfoxide, on the other hand, was too slow to measure withH2SO4. With HCl as the acid, the sulfoxide inversion rate canbe measured but is second order in HCl, so it proceeds by amechanism than involves the formation of R2SCl2 (path B). Adirect comparison of the rates was thus not possible. However,one can estimate that kSe/kS is minimally on the order of 1013 atpH 0, of which roughly 104 can be ascribed to the higherbasicity of the selenoxides, and 109 to the higher electrophilicityof the protonated selenoxides toward attack by water.219

A common effect seen in most heavier−lighter elementcomparisons is a stronger preference for lower oxidation statesin the heavier elements. Selenium is no exception. This effectcan be seen directly in several contexts. For example,selenoxides are able to oxidize sulfides to sulfoxides.220 Asemiquantitative measure of this effect is provided by the

Figure 10. Acyl-transfer equilibrium between a thiol ester and aselenol ester.210

Figure 11. Dipolar nature of chalcogen-oxygen bonds. Y = S, Se.



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[2,3]sigmatropic rearrangement equilibrium between allylsulfoxides and selenoxides, where the equilibrium is slightlyon the sulfoxide side for sulfur and strongly on the selenenateester for selenium. In the specific case shown in Figure 13, this

shift in equilibrium was approximately 13 kcal/mol (the twoequilibria are not strictly comparable since they were measuredat different temperatures). This is one of the largest S/Sedivergences reported.221

Related to this is the divergent behavior of sulfur dioxide andselenium dioxide. SO2 is considered a mild reducing agent,whereas SeO2 is a mild oxidizing agent (Riley oxidation). Bothreagents react with alkenes to form intermediate allylsulfinicand allylseleninic acids (Figure 14). However, the allylsulfinicacid simply reverts to the alkene and SO2,

222 maintaining thehigher oxidation state at sulfur, whereas the allylseleninic acidundergoes [2,3]sigmatropic rearrangement to form the allyl

ester of a divalent selenium species, which rapidly hydrolyzes tothe allyl alcohol.223

The difference in electronic structure of the chalcogen oxidesalso leads to different rates of oxidation and reduction, firstintroduced in Figure 4. Although the first oxidation of sulfides/selenides to sulfoxides/selenoxides is fairly comparable, withselenium being slightly more reactive, the second oxidation isvery much more difficult for selenoxides. This is in part aconsequence of the much higher dipolar character of the Se−Obond, resulting in lower nucleophilicity of the lone pair on Se.In fact, oxidation of sulfides to sulfoxides requires a delicatetouch to avoid overoxidation to the sulfone because the secondoxidation is only a little slower than the first. In comparison,any reasonable oxidants can be used with selenides, since thesecond oxidation to selenones is much slower.Another consequence of the higher dipolar character of

selenoxides versus sulfoxides is that the lone pair on selenium isless nucleophilic than the lone pair on sulfur. Thus, sodiumbenzenesulfinate anion alkylates on sulfur to give the sulfone 19(this is a standard synthetic method for the synthesis ofsulfones), whereas the seleninate anion alkylates on oxygen togive a seleninate ester 20.224

The behavior of these reagents with dienes is similarlyenlightening as SO2 forms the cyclic sulfone 21, whereas SeO2forms the cyclic seleninate ester 22.225 It is believed that theSO2 reaction also proceeds through a cyclic sulfinate ester, butthis rapidly rearranges to the more stable sulfone.226

The redox reactions of thiol/selenols follow a pattern similarto that of the sulfides/selenides. The redox potential of selenolsis much lower compared to that of thiols (−381 mV with DTTas the reductant vs −179 mV with glutathione as the reductantin the context of a glutaredoxin peptide fragment using eitherSec or Cys).227 This means that the equilibrium constant for

Figure 12. Rates of acid-catalyzed inversion of sulfoxides and selenoxides in D2O at 25 °C.

Figure 13. Equilibrium constants between selenoxide-selenenate (−80°C) and sulfoxide-sulfenate in CD2Cl2 at −30 °C.221

Figure 14. Divergent behavior of SO2 and SeO2 toward alkenes.



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the oxidation of a diselenol to a diselenide greatly favors thediselenide. In comparison, the equilibrium constant for thedithiol/disulfide pair was 600-fold lower in this sameexperiment and favors the formation of the dithiol. A similarexperiment conducted not on a peptide fragment but in thecontext of a folded protein (glutaredoxin-3) showed that theratio of equilibrium constants was 8500:1 in favor of thediselenide.228 This experimental system also showed that therate of formation of the diselenide was much faster than therate of formation of the disulfide. While the examples aboveshow that incorporation of Sec in place of Cys results in a verylarge change in redox potential in the context of a peptide orprotein microenvironment, Rozovsky and co-workers haveshown that insertion of Sec does not necessarily confer a largechange in redox potential in the context of a protein.229 Theythusly note that the lower redox potential of a selenosulfidebond 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 isvery little data available for these compounds as they areunstable and undergo rapid disproportionation to diselenides orbecome further oxidized. There are some examples of thesynthesis of stable sulfenic/selenenic acids that make use ofbulky substituents to sterically crowd the chalcogen oxide toprotect it from condensation/disproportionation,230−234 butthere is only one report that makes a direct comparison of thechemical properties of the two so far as we are aware.233 Asignificant finding of this work was that the bond dissociationenergy (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, whichis weaker than a S−H bond (87.6 kcal/mol). This greaterstrength of the O−H bond in the selenenic acid led to a slowerrate of reaction with a peroxyl radical compared to the sulfenicacid.233

Oxidation of selenenic/sulfenic acid results in formation ofseleninic/sulfinic acids. The chemical comparison betweenseleninic and sulfinic acids is analogous to that between SeO2and SO2, or between their hydrates, selenious acid (OSe(OH)2) and sulfurous acid (OS(OH)2). That is, seleninicacids are weak oxidizing agents (in fact seleninic anhydride hasbeen proposed as a useful oxidant for many functionalgroups235−237), whereas sulfinic acids are weak reducing agents.In a striking example of this (eq 1), Kice reported thatPhSeO2H and PhSO2H react with each other to reduce theformer and oxidize the latter (eq 1).238

Seleninic acids are weaker acids than sulfinic acids by about 2pKa units (PhSeO2H 4.79,239 PhSO2H 2.76240), a consequenceof the higher electronegativity and better π-acceptor propertiesof sulfur versus selenium. No information on the basicity ofseleninic acids could be found (pKa of PhSe(OH)2

+). However,one can predict that they would be substantially more basicthan sulfinic acids (in analogy with selenoxides and sulfoxides),and thus acid catalyzed substitutions at selenium would also begreatly 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 reactionprobably involves the sequence of steps outlined in Figure 15.

Benzeneseleninic acid is reduced extremely rapidly by thiols; inone experiment, i-Pr-SH reacted with PhSeO2H in under 30 sat −90 °C (Reich, H. J., Kolonko, K. J., unpublished results),whereas PhSO2H does not detectably react with thiols in weeksat RT.243 The rate of reaction of the seleninic acid form of thesynthetic selenoenzyme selenosubtilisin244 with an aromaticthiol (3-carboxy-4-nitro-benzenethiol) gave an apparent secondorder rate constant of 1.8 × 104 M−1 s−1 (pH 5.0), while that ofan alkaneseleninic acid was ∼200-fold faster (3.3 × 106 M−1

s−1).242 These fast rate constants mean the lifetime of aseleninic acid is on the order of seconds when theconcentration of thiol is in the millimolar range. From suchobservations, one can estimate that the rate of reaction of thiolswith seleninic acids is at least 106 faster than their reaction withsulfinic acids.As discussed above, the reduction of seleninic acid to the −2

and 0 oxidation states is very fast in comparison to the samereduction of sulfinic acids. This represents a point of largedivergence in the chemical reactivity of the respectivechalcogens. The rate of oxidation of seleninic acids to selenonicacids is also a point of divergence in comparison to theoxidation of sulfinic acids to sulfonic acids (noted by Gancarzand Kice238), with the latter being ∼2200-fold faster. At pH 7.1,the rate of oxidation of benzenesulfinic acid is 2.7 × 10−3 M−1

s−1, while that of benzeneseleninic acid is 1.2 × 10−6 M−1 s−1

using H2O2 as the oxidant.243

■ ONE-ELECTRON REDOX REACTIONSFigure 16 shows a pair of reactions in which the one involvingselenium is much slower than the sulfur analog. In a verysignificant paper, Koppenol and co-workers showed that whena thiyl radical is formed in a peptide, Cα−H abstraction isgreatly favored, while the same reaction of a selanyl radical isslow.15,246 The significance of this will be discussed in thefollowing section. The one-electron redox potential of the two

Figure 15. Probable mechanism of the reduction of seleninic acids bythiols.245

Figure 16. Comparison of the rates of intramolecular hydrogenabstraction by thiyl and selanyl radicals.246



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chalcogens is also different; the RS•, RSH/H+ couple has aredox potential of 0.92 V, while the RSe•, RSeH/H+ couple hasa potential of 0.43 V.246 This difference in redox potentialmeans that a thiyl radical is capable of oxidizing tyrosine andtryptophan residues to form the corresponding amino acidradical, while the selanyl radical will not perform the sameoxidation.246

■ WHY SELENIUM? RATE ADVANTAGE VERSUSREDOX ADVANTAGE

A seemingly logical conclusion to the question of “whyselenium?” was arrived at relatively early in the field byreplacement of the catalytic Sec residue of several Sec-containing enzymes with a Cys residue.247−250 The replace-ment of Se with S resulted in mutant enzymes with greatlyimpaired catalytic activity. Conversely, replacement of S with Sein Cys-containing enzymes resulted in enhanced catalyticactivity.228,251−253 Thus, one answer to the question of “whyselenium?” is that the use of Se in oxidoreductases of the typediscussed here provides a catalytic advantage to the enzyme.However, Stadtman made an observation that is oftenoverlooked in the study of selenium in biology. She foundthat the catalytic activity of the Cys-containing selenophosphatesynthetase from E. coli had higher specific activity than the Sec-containing enzyme from H. inf luenzae.254 She noted that “theseresults taken together suggest a role for selenocysteine of H.inf luenzae that is not catalytic.” Kanzok and co-workers alsoconcluded that the use of selenium was not required in theactive site of TrxR because a Cys-ortholog enzyme hadcomparable activity toward its cognate substrate.195 If the use ofSec and other forms of selenium is not entirely related toenhanced catalytic function relative to sulfur, then a secondpossibility is related to selenium’s advantageous redox proper-ties discussed above.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 peroxidaseactivity.244,255−260 This gain of function fits well with itssuperior redox properties relative to sulfur described above.The functional definition of a peroxidase is outlined in Figure17. Selenium confers peroxidase activity to an enzyme becauseit is both a good nucleophile and a good electrophile. Thisproperty allows it to easily cycle between reduced and oxidizedstates without becoming permanently oxidized. In this respect,

the redox properties of selenium more closely resemble atransition metal in comparison to sulfur.The gain of peroxidase activity conferred by selenium led to

the hypothesis that selenium confers resistance to irreversibleoxidation and inactivation due to the difference in redoxchemistry between selenium and sulfur shown in Figure 4.12,263

This hypothesis is a late manifestation of an idea that has beenexpressed in other forms, or went unrecognized by researcherswho did not realize the significance of their data. Thishypothesis is reviewed below.Chaudiere and co-workers 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 ofGpx1, which had much lower activity than the Sec-containingGpx1 but was readily inactivated in the presence of its substrate,H2O2, and organic hydroperoxides. Chaudiere hypothesizedthat the Cys-mutant Gpx1 was inactivated due to oxidation ofthe peroxidatic Cys residue to Cys-SO2

− with possible β-elimination to form dehydroalanine. This hypothesis wasperhaps validated several decades later by Bellelli and co-workers who crystallized the Cys-mutant of Sec-containingGpx4 from Schistosoma mansoni.264 This X-ray crystal structureof the Gpx4 mutant showed the presence of a sulfonic acidresidue (see Figure 18).264 The Cys-mutant of S. mansoni Gpx4was inactive at all stages of purification, even though reducingagents were present. One interpretation of these data thatmatches Chaudiere’s observation is that the Cys-mutantenzyme was able to react with the oxidant, but the S-oxidethat formed was not electrophilic enough to be resolved back to

Figure 17. Peroxidase cycle for Sec- and Cys-peroxidases. Both types have a nucleophilic selenolate/thiolate that reacts with an oxidant to form aselenenic/sulfenic acid, which is then resolved by another thiol to form a mixed selenosulfide/disulfide. The active enzyme is regenerated by theaddition of a second mole of thiol. The 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 themechanism, 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 theabsence of selenium using a combination of these properties.261 One large difference is the inability of the Cys-SO2

− form of the Cys-peroxidases toreturn to the active catalytic pathway by reduction with exogenous thiol, while reduction of the Sec-SeO2

− form is possible for Sec-peroxidases asshown by the work of Hilvert et al.242 Peroxiredoxin is one exception to this rule, but it requires a special repair enzyme to reduce the Cys-SO2

− formof the enzyme,262 while the reduction of Sec-SeO2

− of Sec-peroxidases can be easily reduced by ascorbate and glutathione.

Figure 18. (A) A close-in view of the catalytic triad of S. mansoni Gpx4consisting of Cys43 oxidized to a sulfonic acid, Gln78, and Trp132(PDB 2v1m). (B) A close-in view of the catalytic triad ofselenosubtilisin showing Sec221 in the seleninic acid form alongwith Asp32 and His64 (PDB 1sel). The selenium atom is coloredmagenta.



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the active form of the enzyme. This results in overoxidation andinactivation of the Cys residue of the mutant enzyme.Very recently, Ursini and co-workers presented evidence that

Sec-containing rat Gpx4 avoids overoxidation of the Sec residueby using a backbone nitrogen to attack the selenenic acidintermediate to form an eight-membered ring selenenamidewhen glutathione becomes limiting.265 Formation of theselenenamide is thought to be a type of safety mechanismthat 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 themutant became irreversibly oxidized to the sulfonic acid form.The experimental evidence of selenenamide formation in thenative protein fits well with earlier work by Reich and co-workers who synthesized model compound 23, which becameoxidized to both the selenenamide 24 and seleninamide 25 inthe presence of an oxidant.266 Selenenamides have theinteresting property of being significantly more basic (3.4 pKunits) than the corresponding sulfenamides.267 This propertyshould greatly enhance opening of the ring by an attackingthiol. This is yet another large chemical difference betweensulfur and selenium.

This story continues in 1993 when Hilvert and co-workersdiscovered that selenosubtilisin, a semisynthetic selenoen-zyme,244 was not inactivated by oxidation to the Enz-Sec-SeO2

− form as the enzyme could regain activity by the additionof two additional moles of thiol.242 There is very strongevidence that selenosubtilisin is capable of existing in the Enz-Sec-SeO2

− form as supported both by X-ray crystallography268

(see Figure 18B) and 77Se NMR spectroscopy.269 At the time,the importance of this discovery as it relates to irreversibleoxidative inactivation was unclear since little was then knownabout the existence of the corresponding sulfinic acid form inproteins. This is not the only example of the seleninic acid formin an enzyme. Branlant and co-workers converted glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) into a selenoen-zyme by producing the protein in a cysteine auxotroph andreplacing cysteine in the media with selenocysteine.256

Replacement of Cys in GAPDH with Sec results in a gain ofperoxidase activity, but more importantly it showed that theenzyme existed in the Enz-Sec-SeO2

− form (by the use of massspectrometry) without resulting in inactivation.256 It is known,however, that oxidation of GAPDH to the Enz-Cys-SO2

− formresults in permanent oxidative inactivation.270 While theseexamples show that the seleninic acid form of a selenoenzymecan be part of a catalytic cycle, there is no evidence as of yetthat 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 ofenzymes was oxygen tolerant,271 unlike [Fe−Fe]-hydrogenasesand most [NiFe]-hydrogenases. Maroney and co-workersprovided evidence that selenium confers oxygen tolerance in[NiFeSe]-hydrogenases by synthesizing nickel-containingmimics of the active site that had either sulfur or seleniumligands as shown in Figure 19.272 Reaction of the thiolatocomplex with molecular oxygen resulted in a monosulfinato

complex. In striking contrast, the selenolato complex resistedoxidation. In follow up work, Maroney comments thatsubstitution of selenium for sulfur in the [NiFeSe]-hydrogenasefrom D. baculatum (a strict anaerobe) makes “...the enzymemore stable to oxidation and may be isolated in air in a statethat does not require reductive activation.”273 This view ofselenium in an enzyme strongly diverges from the earliest viewof the use of selenium in an enzyme expressed by Bock: ‘‘UGAwas originally a sense codon for Sec in the anaerobic world,perhaps 2 to 3 billion years ago, and after introduction ofoxygen into the biosphere this highly oxidizable amino acidcould be maintained only in anaerobic organisms or in aerobicsystems which evolved special protective mechanisms.”162

Based on the results of Maroney, it is tempting to speculateon the exact opposite scenario: The use of Sec in proteinsreached a maximum during the oxygen peak of the Permianperiod and then declined as the amount of oxygen in theatmosphere declined.In a followup to the work by Maroney more than a decade

later, Armstrong and co-workers performed a study of the[NiFeSe]-hydrogenase from D. baculatum using protein filmvoltammetry.274 The goal of the study was to more completelycharacterize the A and B states of the enzyme, with the A statecorresponding to an “Unready” form (formed upon reactionwith O2), while the B state corresponds to the “Ready” form.The “Ready” and “Unready” states are so named because oftheir kinetic characteristics with respect to reactivation of theenzyme (slow and fast, respectively). An important finding ofthis work was that the enzyme was capable of hydrogenaseactivity in the presence of 1% O2, and the “Unready,” A-form ofthe enzyme was reactivated 100-fold faster compared to thenonselenium containing A-form of the [NiFe]-hydrogenasefrom A. vinosum. Armstrong contemplated that the inactive A-form is due to the formation of a Se-oxide that is rapidlyreduced to the active form of the enzyme.274

The role of selenium in conferring oxygen tolerance to[NiFeSe]-hydrogenases was further addressed by Matias andco-workers who solved the X-ray crystal structure of the[NiFeSe]-hydrogenase from D. vulgaris.275 The electron densitymap of the [NiFeSe] complex was interpreted in terms of threestructures in a 70:15:15 ratio, respectively, as shown in Figure20. Structures I and II were persulfurated, while the third had adirect Ni−Se linkage. Their analysis revealed that selenium in Iand II shields the nickel atom from small molecules such asmolecular oxygen, preventing inactivation from the formationof an oxo ligand at the nickel center. This may be due toselenium’s ability to form strong metal bonds, as it does withmercury.202 However, the presence of the Sec residue in theenzyme does not prevent other oxidation events such as theconversion of Cys75 from a nickel sulfide to a nickel sulfinate.

Figure 19. Reaction of thiolato or selenolato nickel complexes withmolecular oxygen. Only the sulfanato complex resulted in oxidation.272



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Another example of selenium conferring resistance tooxidation comes from a Sec for Cys substitution in CYP119,a cytochrome P450 heme-containing monooxygenase fromSulfolobus acidocaldarius.276 Oxidation of SeCYP119 with excessm-chloroperbenzoic acid (mCPBA) led to the presence of anew oxidized species that absorbed at 406 nm in a magneticcircular dichroism spectrum. The identity of this oxidizedspecies could not be elucidated, but this oxidized form could bereduced back to the original state in the presence of a reducingagent such as DTT. Importantly, the introduction of seleniuminto the enzyme protected against heme destruction comparedto the protein containing sulfur (either selenium or sulfur areligands for the iron center of the heme), where significantlymore heme destruction occurred (Figure 21).The most recent evidence for the hypothesis that selenium is

used to prevent irreversible oxidative inactivation comes fromHondal’s laboratory, who tested this hypothesis in Sec-containing thioredoxin reductase (TrxR). The approach wasto compare the ability of the Sec-containing TrxR to resistoxidative inactivation compared to a Cys-ortholog from D.melanogaster (DmTrxR).263 DmTrxR was used for directmechanistic comparisons because it has similar enzymearchitecture to the mammalian Sec-containing enzyme, butCys replaces Sec.195,277 DmTrxR is 50% inactivated at 1 mMH2O2, while the Sec-TrxR retained virtually all of its activity asshown in Figure 22. To support the hypothesis that seleniumconfers the ability to resist oxidative inactivation to an enzyme,Cys was replaced with Sec using a sophisticated proteinengineering technique called expressed protein ligation,278 andthen the ability of the mutant enzyme to resist H2O2-mediatedinactivation was measured by assaying for the remainingactivity. As shown in Figure 22, replacement of a single atom inthe enzyme, selenium for sulfur, enabled resistance to oxidativeinactivation by H2O2,

263 identical to the examples discussedabove. Because of this effect, we call this the “Sec-rescue”-TrxR.Both the Sec-TrxR and the Sec-rescue-TrxR were also able toresist inactivation by one-electron oxidants such as the hydroxylradical as had been predicted by Steinmann and co-workers,15

but Cys-containing DmTrxR could not.

■ CONCLUSIONAlmost all chemical reactions involving selenium are faster incomparison to the same reaction with sulfur. For this reason, itis tempting to conclude that nature chose selenium to replacesulfur for this enhanced chemical reactivity in order toaccelerate enzymatic reactions. In contrast, our answer to thisquestion is that nature has chosen selenium due to its uniqueability to react with oxygen and related ROS in a readilyreversible manner. Both sulfur and selenium are goodnucleophiles that react with ROS in two-electron oxidationevents and, in so doing, become oxidized. The S-oxides and Se-oxides that are formed in this process show very strongdivergence in their chemical reactivities, due in large part tovery weak π-bonding in the Se-oxide. As a result, Se-oxides havea much stronger ability to be rapidly reduced back to theoriginal state in comparison to S-oxides. The ability of seleniumto both rapidly become oxidized and then be rapidly reducedhas been referred to as the “selenium paradox.”12 Evidence forthis “selenium paradox” comes from the gain of function thatselenium confers to both natural and artificial selenoenzymes;selenium confers resistance to inactivation by oxidation as thenumerous examples discussed above show.Closely related to the reversibility of two-electron oxidation

events is the enhanced stability of the selenayl radical comparedto the thiyl radical.15,246 This means that selenium-containingproteins are much better able to withstand one-electronoxidation events.263 Both of these hypotheses fit well with

Figure 20. Three structures of the NiFe center in the [NiFeSe]-hydrogenase from D. vulgaris.275 Structure I corresponds to theoxidized form, while structure III corresponds to the reduced form.Structure II is an intermediate between I and III.

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, doublearrows), but not so the selenium-containing version. An unidentified, oxidized form of Sec could be reduced back to the parent compound byaddition of dithiothreitol (DTT) as indicated, but the oxidized form of Cys could not.

Figure 22. (bar graph left) Activity of TrxR enzymes after H2O2exposure. The blue bar is the activity of the Cys-TrxR (DmTrxR). Thered bar is mouse Sec-TrxR, and the green bar is the Cys → Sec mutantof DmTrxR. In this experiment, the enzymes were first exposed toH2O2. Then, the H2O2 was quenched with catalase, and substrate(Trx) was added. Then, the amount of activity remaining wasmeasured. Reprinted with permission from ref 263. Copyright 2013American Chemical Society. (top right) Depiction of the gain offunction that Sec confers to an enzyme in the “Sec-Rescue”-TrxR-resistance to oxidative inactivation.



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the idea that oxygen in the biosphere is perhaps the strongestevolutionary force in the history of life on Earth,279 and thechemistry of selenium is ideally suited to respond to one- andtwo-electron oxidation events.

■ AUTHOR INFORMATIONCorresponding Authors*Tel.: (608) 262-5794. E-mail: [email protected].*Tel.: 802-656-8282. Fax: 802-862-8220. E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors would like to thank Michael Maroney of theUniversity of MassachussettsAmherst, Paul Copeland ofRutgers University, and Raymond F. Burk of VanderbiltUniversity (emeritus), for providing critical commentary andencourgement. The authors would also like to thank StephenEverse of the University of Vermont and Francessco Angelucciof the University of L’Aquila for help in constructing figures.

■ DEDICATIONThis paper is dedicated to Raymond F. Burk on the occasion ofhis recent retirement from Vanderbilt University and for hismany years of dedication to the study of selenium biochemistry.

■ KEYWORDSSelenocysteine: The selenium analog of cysteine. It is the21st amino acid in the genetic code.Selenouracil: The selenium analog of thiouracil. Boththiouracil and selenouracil are modified bases found in theanticodon loop of tRNA molecules.Selenoxide elimination: A chemical method for generatingcarbon−carbon double bonds that involves abstraction of ahydrogen that is beta to the selenoxide. This reaction is veryfast and is perhaps the single most important use of seleniumby synthetic organic chemists.Selenenic acid: The selanyl mono-oxide form of selenium. Itis the selenium analog of sulfenic acid.Seleninic acid: The selanyl dioxide form of selenium. It isthe selenium analog of sulfinic acid.Selenonic acid: The selanyl trioxide form of selenium. It isthe selenium analog of sulfonic acid.Selenoxide: The monooxidized form of a selenide. It is theselenium analog of a sulfoxide.Selenone: The dioxidized form of a selenide. It is theselenium analog of a sulfone.Selanyl radical: The radical formed on selenium when aselenol loses a hydrogen atom. It is the selenium analog of athiyl radical.Oxidative inactivation: The process by which an enzymeloses activity due to oxidation of a functional groupimportant for catalytic activity.

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Why Nature Chose Selenium - UW-Madison Chemistry · Why Nature Chose Selenium Hans J. Reich*, ‡...

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