Review Article

Redox chemistry and chemical biology of H2S, hydropersulfides,and derived species: Implications of their possible biological activityand utility

Katsuhiko Ono a, Takaaki Akaike b, Tomohiro Sawa b, Yoshito Kumagai c, David A. Wink d,Dean J. Tantillo e, Adrian J. Hobbs f, Peter Nagy g, Ming Xian h, Joseph Lin i, Jon M. Fukuto a,n

a Department of Chemistry, Sonoma State University, Rohnert Park, CA 94928, USAb Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japanc Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japand Tumor Biology Section, Radiation Biology Branch, National Cancer Institute, Bethesda, MD 20892, USAe Department of Chemistry, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USAf William Harvey Research Institute, Bart & London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UKg Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungaryh Department of Chemistry, Washington State University, Pullman, WA 99164, USAi Department of Biology, Sonoma State University, Rohnert Park, CA 94928, USA

a r t i c l e i n f o

Article history:Received 16 July 2014Received in revised form2 September 2014Accepted 4 September 2014Available online 16 September 2014

Keywords:Hydrogen sulfidePersulfidesPolysulfidesThiolsThiol redox

a b s t r a c t

Hydrogen sulfide (H2S) is an endogenously generated and putative signaling/effector molecule. Despiteits numerous reported functions, the chemistry by which it elicits its functions is not understood.Moreover, recent studies allude to the existence of other sulfur species besides H2S that may play criticalphysiological roles. Herein, the basic chemical biology of H2S as well as other related or derived species isdiscussed and reviewed. This review particularly focuses on the per- and polysulfides which are likely inequilibrium with free H2S and which may be important biological effectors themselves.

& 2014 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Biological chemistry of H2S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Basic chemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Interaction of H2S with metals (iron hemes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84H2S and RSSH formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Alkyl hydropersulfide formation, chemistry, and biological relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85RSSH biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Alkyl hydropersulfide chemistry: Nucleophilic and electrophilic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86One-electron redox chemistry of persulfides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Two-electron redox chemistry of hydropersulfides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Chemistry of dialkylpolysulfides and alkyl hydropolysulfides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Contents lists available at ScienceDirect

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Free Radical Biology and Medicine

http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.0070891-5849/& 2014 Elsevier Inc. All rights reserved.

Abbreviations: sGC, soluble guanylate cyclase; NHE, normal hydrogen electrode; MbFeIII, methemoglobin; HbI, H2S-binding hemoglobin; MPO, myeloperoxidase; HbFeII–O2,oxyhemoglobin; sulfHb, sulfhemoglobin; sulfMb, sulfmyoglobin; CcO, cytochrome c oxidase; SQR, sulfide:quinone oxidoreductase; CSE, cystathionine γ-lyase; CBS,cystathionine β-synthase; Trx1, thioredoxin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 8-NO2-cGMP, 8-nitro-cyclic guanosine monophosphate; BDE, bonddissociation energy; NHE, normal hydrogen electrode

n Corresponding author. Fax: þ1 707 664 3378.E-mail address: fukuto@sonoma.edu (J.M. Fukuto).

Free Radical Biology and Medicine 77 (2014) 82–94

Comparison of persulfide chemistry with peroxide chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Possible biological implications of persulfide and polysulfide generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Introduction

Hydrogen sulfide (H2S) has been reported to be an important,endogenously generated, small-molecule signaling agent withnumerous physiological functions (for recent reviews, see [1–4]).It is proposed to be one of the so-called “gasotransmitters”1 alongwith nitric oxide (NO), carbon monoxide (CO), dioxygen (O2), andderived species (for example, [5,6]). Although the reported func-tions of H2S are numerous, the chemical mechanisms associatedwith its biological activity are not established. Moreover, the actuallevels of H2S present in biological tissues and fluids have been amatter of some contention and controversy [7,8] with earlyreports of levels as high as 20–80 μM in plasma. However, it isbecoming increasingly clear that concentrations of “free” H2S arelow (submicromolar) but that there is a labile “pool” of sulfur-containing species, in equilibrium with free H2S, that can liberateH2S [9]. The fact that there is this large labile pool of H2S-releasingspecies indicates likely biological significance to these H2S pre-cursor molecules (vide infra).

As with all biological signaling species, the utility of the small-molecule signaling agents is due to their unique chemistry andphysical properties. That is, the signaling properties of thesespecies are due to their specific/selective chemical interactionswith biological targets and subsequent effects on the function ofthese target molecules (for example, [10]). The best and mostestablished example of this (at least among the small-moleculesignaling agents) is the interaction of NO with its primarybiological target, the enzyme-soluble guanylate cyclase (sGC).The regulatory heme of sGC binds NO, which presumably resultsin the loss of an iron heme axial histidine ligand leading toactivation of the enzyme. Significantly, NO appears unique in itsability to elicit this type of chemistry (dissociation of the axialligand on NO binding [11]). Unlike NO, the exact nature of thechemistry associated with the biological activity of H2S is notfirmly established. However, considering the fact that H2S is firstand foremost a simple thiol, it may be expected that the chemicalbiology of H2S involves, at least in part, its properties as anucleophile/reductant which can lead to changes in sulfur oxida-tion state. After all, much of the biological utility of thiols relies onthese properties and interaction with other thiol species or metals.Thus, this review will focus on the biological chemistry of H2S (andmore importantly, species derived from H2S or possibly those thatcan serve as sources of H2S such as enzyme-mediated persulfideformation, vide infra) as a means to begin to understand itsbiological function and utility.

Although one of the focuses of this review is H2S chemicalbiology, readers will note that the majority of the discussionpresented herein relates to other sulfur species. This may seemcurious considering the title of this review and the prevalence ofthe recent literature that mostly focuses primarily on H2S. There-fore, before continuing further, it is worth presenting one of thetenets of this review: Some of the biological signaling/effects

commonly associated with H2S may be due to other sulfur-containing species which can degrade to release H2S (makingH2S a possible marker for these species). In studies involvingpharmacological or experimental addition of H2S (or H2S donors),the primary effector molecule may also not be H2S, but ratherthese same species made via reaction of exogenous H2S withoxidized thiol precursor species (vide infra). This is, of course,speculative at this time but justifies the time spent discussingother chemical species besides H2S. This is not to say that H2S isbiologically innocuous. On the contrary, it seems likely thatbiological H2S generation has function. What is being consideredherein, however, is that other sulfur-containing molecules haveunique and biologically important properties that allow them tobe specific effector/signaling species. The fact that they mayrelease H2S under certain conditions may be biologically relevantsince, considering the parsimony of Nature, it seems probable thatH2S release is also purposeful. Regardless, discussions of thechemistry of many of the sulfur species besides H2S are given toprovide the basis of future work/discussion of this signaling.

Nomenclature

As with many fields, unwieldy, inappropriate, or inconsistentterminology can be a major obstacle to understanding the chem-istry being described. This seems especially true when describingthe chemistry/biology of sulfur-containing molecules. For exam-ple, the terms thiol, sulfhydryl, and mercaptan have been used todenote essentially the same functional group, -SH. Although thereare published rules or guidelines for the naming of sulfur-containing molecules (for example, [12]) they often do not coverall the possible sulfur-containing species and many of these rulesare not rigorously followed or are cumbersome. To be sure, thisreview is not intended to serve as a treatise of sulfur nomencla-ture. However, it is worthwhile to define the terms that will beused herein to avoid confusion. Consistent with most of thecurrent literature, the terms hydrogen sulfide, thiol, and disulfidewill be used to describe H2S, RSH, and RSSR, respectively. Hydro-sulfide and sulfide are often used to specifically denote the anionicspecies HS� (i.e., sodium hydrosulfide, NaHS) and S2� (i.e., sodiumsulfide, Na2S). When the general term hydrogen sulfide is used,especially pertaining to its presence in biological systems, this willinclude all protonation states (H2S, HS� , and S2�). Confusion canoccur when referring to multisulfur species. The term alkylhydropersulfide denotes a molecule whereby a disulfide is sub-stituted on one end with an alkyl group and the other end with ahydrogen atom (RSSH). The term “alkyl hydrodisulfide” could havebeen used here (consistent with the naming of RSSR a disulfide)but since it is important to draw some structural and chemicalanalogy to peroxides (dialkyl peroxide, ROOR; alkyl hydroperox-ide, ROOH; and hydrogen peroxide, HOOH) the term “persulfide”was selected (note that “perthiol” can also be used here, but forthe sake of consistency this term will not be used; however, whenreferring to the radical RSS � , “perthiyl” is used). The term alkylhydropolysulfides is used to describe species of the general formRSSnH (n41). When referring to a specific polysulfide, the numberof sulfur atoms can be indicated (e.g., RSSSH can be referred to asan alkyl hydrotrisulfide). A similar system for dialkyl polysulfides

1 The term “gasotransmitter” is a poor description of these small-moleculesignaling agents since it misrepresents their chemical state when they are actingbiologically. They do not exist as gases, but as solutes when they are acting asphysiological effector/signaling agents. This term is only noted here due to itsunfortunate prevalence in the current literature.

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will be adopted herein: dialkyl polysulfides will be generallyreferred to as RSSnR (n41) and specific dialkyl polysulfides suchas RSSSR will be termed dialkyl trisulfide. Especially problematicare the oxidized forms of hydropersulfides and hydropolysulfides.For example, RSSOH cannot be named a “persulfenic acid” sincethis term has been used to describe a molecule of the type RSOOH.In order to distinguish between RSSOH and RSOOH, a species ofthe type RSSOH will be referred to herein as an alkyl perthiosul-fenic acid. Due to the possible confusion and ambiguity associatedwith adopting any convenient nomenclature scheme, chemicalformulas will be given as much as possible.

Biological chemistry of H2S

Basic chemical properties

Herein, only a brief review of fundamental H2S chemistry is given.Other, more comprehensive reviews are available (for example,[13–15]). Unlike the other small-molecule signaling agents NO, CO,or O2, H2S is ionizable. The pKa of H2S is 6.8 and the pKa of HS� isapproximately 14. Thus, at physiological pH, the monoanion HS� isthe predominant species with negligible concentrations of S2� . ThepKa values for many thiols (RSH) are significantly higher than that ofH2S (although there are numerous thiols in proteins that have verylow pKa values). Therefore, H2S generally has a higher equilibriumconcentration of the anionic species, HS� , compared to most “free”thiols. The reduction potential for HS � (the thiyl radical) is reported tobe approximately 1 V (pH 7, vs a normal hydrogen electrode (NHE))[16,17]. Therefore, H2S is not a particularly good one-electron reduc-tant and the oxidized thiyl species, HS � , is a good oxidant. Overall, theone-electron chemistry of HS�/HS � does not appear to be significantlydifferent from that of normal biological thiols/thiyls. The one chemicalproperty that distinguishes H2S from other biologically relevant alkylthiol species (such as glutathione (GSH) or cysteine (Cys–SH)) is thefact that it has two dissociable protons that can be readily substitutedwith other atoms/functional groups (alkyl thiols, on the other hand,have only one dissociable proton). This is an important property sinceit allows H2S to form bridges between atoms (i.e., thioethers (RSR),metal sulfides (metal–S–metal). Moreover, this property of H2S alsoallows for the formation of per- and polysulfides (i.e., RSnH, n41 andRSnR, n42). The prevalence, relevance, and chemical properties ofthese polysulfur species will be discussed in more detail later.

Interaction of H2S with metals (iron hemes)

A major target for H2S biology/toxicology has been proposed to bemetals. As with the other established small-molecule signaling agents(i.e., NO, CO, O2, and derived species) whose primary biologicalreceptors are likely metalloproteins, H2S as well as hydropersulfides(vide infra) have the ability to interact with metalloproteins and theseinteractions may also be important to their biological function.Although there are numerous metals (i.e., copper, iron, manganese,etc.) and types of metalloproteins (iron–sulfur proteins, nonheme ironproteins, zinc fingers, etc.), the emphasis herein will be on iron hemeproteins. The decision to focus primarily on this class of metallopro-tein is not because the other types are not biologically relevant butrather more due to the fact that the interactions of H2S (and relatedspecies) with iron heme proteins have been the most studied (albeit,somewhat sparsely).

One of the earliest studies of the interaction of H2S with a hemeprotein is by Keilin [18] where it was demonstrated that thereaction of H2S with methemoglobin (MbFeIII) resulted in aspectral change caused by a presumed reversible generation ofan H2S–MbFeIII adduct. Much more recently, it was shown thatother ferric heme proteins can react directly with H2S to give an

FeIII–SH complex with the same spectral changes observed in theinitial study by Keilin [19,20]. One of the most studied of all H2S–heme protein interactions is that of H2S with a hemoglobin of thebivalve mollusc, Lucina pectinata, that inhabits H2S-rich environ-ments (for example, [21–26]). Interest in this specific interactionstems from the fact that L. pectinata possesses a specialized H2S-binding hemoglobin (HbI) used to transport H2S to symbioticchemoautotrophic bacteria living in its gills. These bacteria utilizeH2S to fix CO2 into hexoses that are subsequently used by themollusc. Based on X-ray crystallographic and mutation studies, itappears that stable H2S binding to ferric HbI is facilitated byseveral interactions between the bound H2S and the specific distalresidues—hydrogen bonding between a glutamine carbonyl andbound H2S, a hydrophobic “cage” made up of phenylalanines andfavorable electrostatic interactions between phenylalanines andthe H2S ligand (for example, [23,27]). Moreover, the nonpolarbinding pocket appears to disfavor deprotonation of the ferric H2Scomplex, contributing to its stability [26].

Based on the discussion above, heme proteins can be designedto reversibly bind/carry H2S. However, the interaction of H2S withother ferric heme proteins or model porphyrin systems (notspecially designed to bind H2S) can undergo further chemistryafter initial H2S binding. For example, Pietri and coworkers [26]report that mutation of the important nonpolar residues in theferric HbI binding site to polar residues can facilitate deprotona-tion of the bound H2S ligand to form an FeIII–HS� complex, whichcan result in ferric to ferrous reduction. It was also proposed thatthis reduction was facilitated by excess H2S (Reaction (1),HbIm¼mutated HbI).

HbIm�FeIII�H2S-[HbIm�FeIII�HS�’-HbIm�FeII�HS � ]þH2S--HbIm�FeII (1)

The mechanism by which excess H2S facilitates reduction of theferric–H2S complex (or ferric–HS� complex) is intriguing and hasrecently been demonstrated for myeloperoxidase (MPO) usingspectroscopic measurements on the FeII–sulfide complex, whichwas also independently synthesized by the reaction of reducedMPO with sulfide [20]. The sulfur-containing product of thisreaction has been proposed to be dihydrogen persulfide (H2S2)[26]. A recent study by Pavlik and coworkers [28] examined thereaction of H2S with model ferric and ferrous porphyrins andfound that HS� readily binds ferrous species and reduces ferricporphyrins (although the possible role of excess H2S was notspecifically addressed). This result is consistent with the idea thatH2S binding/carrying ferric porphyrins require special ligandbinding pockets to accommodate H2S binding and avoid HS�

binding/formation and subsequent reduction.One of the earliest reports of a direct reaction between H2S and

heme proteins was the finding that exposure of oxyhemoglobin(HbFeII–O2) to H2S resulted in a green pigment ([18] and referencestherein). It was later found that the green pigment was an H2S-modified heme referred to generally as sulfhemoglobin (sulfHb) andcould only be generated when hemoglobin was exposed to bothH2S and O2 (the same process for myoglobin was also observed,making sulfmyoglobin (sulfMb)) (for a review, see [29]). Thegeneration of sulfHb or sulfMb is considered irreversible anddeleterious since the H2S-mediated modification of the porphyrinlowers the O2 binding affinity of both proteins [30]. The mechanismof formation of the sulfHb/sulfMb is currently unknown butthought to involve reaction of H2S with a high-valent iron-oxospecies leading eventually to covalent incorporation of a sulfuratom into the pi system of one of the pyrole rings of the heme[29,30]. To be sure, formation of sulfhemes is not restricted tohemoglobin and myoglobin, as other proteins such as catalase andlactoperoxidase can also form sulfhemes when exposed to H2S [29].

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Since H2S is generated endogenously (as a presumed signalingspecies made by mammalian cells and by colonic bacteria), theformation of sulfhemes may be prevalent under normal (nonpatho-logical) conditions. Whether these basal levels of sulfheme proteinshave physiological function remains to be determined.

From a purely toxicological perspective, the interaction of H2Swith cytochrome c oxidase (CcO) has been of great interest sinceH2S toxicity is generally thought to be due, at least in part, toinhibition of mitochondrial respiration. Indeed, inhibition of CcOactivity is proposed to be a sensitive biomarker for H2S exposure[31]. However, a watershed paper from the Roth lab [32] reportedthat 80 ppm exposure of mice to H2S put them in a state ofsuspended animation akin to a hibernative state. This state ofsuspended animation was presumably due to an inhibition of CcO.Significantly, cessation of H2S exposure led to full metabolicrecovery with no measureable deficit. The Roth group also showedthat placement of mice into an H2S-mediated state of suspendedanimation also protected them from lethal hypoxia [33]. Thus,exposure of mice to pharmacological levels of H2S that presumablyinteract with CcO can have nontoxic and even beneficial effects. Forthese reasons, examination of the interaction of H2S with CcO maybe important in the elucidation of H2S-mediated biological activity.

Although H2S can clearly inhibit CcO, this effect is complex andhighly dependent on the levels of H2S (for example, [34]). Forexample, the mitochondria of marine invertebrate living in anH2S-rich environment can utilize H2S at low levels (o20 μM) asan electron source for oxidative phosphorylation [35]. Moreover,this same phenomenon can be observed in chicken liver mito-chondria (at H2S levels o5 μM) [36] and in mammalian cells (atlow μM concentrations of H2S) [37]. In mammalian cells, it isproposed that H2S can reduce a mitochondrial element (e.g.,coenzyme Q) between complexes I and III via the actions ofsulfide:quinone oxidoreductase (SQR) (vide infra). However, athigh concentrations of H2S, inhibition of mitochondrial activity isobserved, possibly via interaction at a second mitochondrial site(likely CcO) [37]. A recent study examining the interaction of H2Swith CcO by Collman et al. [38] utilizes a model system consistingof a synthetic molecule that replicates the iron heme–copper O2

binding site of CcO. Importantly, this model system avoids inter-actions of H2S with other mitochondrial components. It was foundthat H2S rapidly reduces the oxidized ferric or cupric species ofCcO to their active ferrous and cuprous, O2-binding forms. Thisindicates that H2S can serve as a source of electrons for CcO-mediated O2 reduction. It appears that low levels of H2S will noteffectively compete with O2 binding. However, at high levels ofH2S, it can compete with O2 binding, thus inhibiting CcO activity.These results are consistent with the idea that H2S can supportCcO activity at low concentrations since it can serve as reducingequivalents but at high concentrations can compete with O2 at theferrous–cuprous O2-binding site and inhibit CcO activity. A recentreview by Pietri et al. [30] proposes a slightly more detailedexplanation whereby at low H2S concentrations the polar environ-ment of the heme a3 center (the heme center that is normallyinvolved with O2 binding) promotes heme a3 reduction. However,at higher levels of H2S, reduction of the CuB center (also involvedin O2 binding) occurs resulting in the formation of a stable,reduced CuB–H2S complex as well as an unstable inhibitory FeII–H2S complex of heme a3. With an eventual exhaustion of H2S,formation of a stable FeIII–H2S complex inhibits the system.Regardless, it is clear that the inhibition of CcO is complex, H2Sconcentration dependent, and involves numerous reactive sitesand intermediate species.

Based on in vitro, isolated mitochondria and purely chemicalstudies, it is clear that the effect of H2S on mitochondrial function(and CcO activity) is at least biphasic and potentially complex. Whatis also clear is that high levels of H2S are potentially deleterious with

regard to inhibition of mitochondrial respiration. In mammalian cells,levels higher than low micromolar may adversely affect mitochon-drial activity. This, of course, would predict that the extremely highlevels of H2S originally reported (levels as high as 100 μM) are likelyincorrect (for example, [39]), and if anything, reflects the large size ofan H2S-releasing pool [9], a significant part of which could representpersulfide/polysulfide compounds recently found to be at highphysiological concentrations [40] (vide infra).

H2S and RSSH formation

H2S is a fully reduced sulfur species with a formal oxidationstate of 2–. As such, in biological systems, it can only be oxidized.Since both thiols (RSH) and H2S are formally at the same oxidationstate, they will not react with each other. Thus, fully reduced thiolsare not expected to be direct targets of H2S reactivity [41].However, oxidized thiol species such as dialkyl disulfides (RSSR)or sulfenic acids (RSOH) do react with H2S. For example, H2S hasbeen shown to react with disulfides to give the correspondingthiol and RSSH (Reaction (2)) (for example, [42,43]).

H2SþR0SSR⇌R0SSHþRSH (2)

As noted above, the existence of H2S in biology allows for thegeneration of RSSH (since thiols can only make RSSR via analogouschemistry). The reaction of H2S with a dialkyl disulfide can beviewed simply as a reaction of a nucleophile, H2S/HS� , with anelectrophile, RSSR analogous to the classical cysteine thiol/disul-fide exchange reactions [44]. Although the equilibrium constantfor Reaction (2) is not known and likely to be highly dependent onthe nature of “R” and reaction conditions (e.g., pH), the possibilityof this reaction having biological relevance has been reported [43].To be sure, it has also been stated that the reaction of H2S withoxidized sulfur species like RSSR is not likely to occur due to thelow concentration and relatively unfavorable reduction potentialfor H2S and competition with other thiol species (for example,[45]) (it should be noted, however, that the idea that an unfavor-able reduction potential opposes this reaction has been questioned[13]). However, the reactivities of RSSR moieties toward thiolnucleophiles vary on a large scale [44] and therefore it wouldnot be unreasonable to expect that biological H2S exists in redoxequilibrium with other reduced and oxidized thiol species (asdepicted in Reaction (2), although the positions of these equilibriaare not known and may vary greatly depending on the oxidizedthiol species. Thus, the relevance of Reaction (2) in formingbiological persulfides remains to be established.

Alkyl hydropersulfide formation, chemistry, and biologicalrelevance

RSSH biosynthesis

It is intriguing to imagine that the biological actions of H2Scould be, to a significant degree, the result of interactions withoxidized thiols in proteins or peptides. That is, reaction of H2S withan oxidized thiol species (RSSR, RSOH) associated with a protein orpeptide results in the generation of an RSSH species in the protein/peptide, which may be the actual biological effector species.Importantly, biological RSSH generation can occur enzymaticallyto give directly an RSSH species, which can then go on to set upRSSH/H2S equilbria (Reaction (2)) (both or either of which may beimportant signaling species). For example, cystine (Cys–SS–Cys)can be converted to cysteine hydropersulfide (Cys–SSH) viathe actions of cystathionine γ-lyase (CSE) [46] or cystathionineβ-synthase (CBS) [40] (Fig. 1).

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–94 85

Interestingly, the Km for the reaction of Cys–SS–Cys with CSE isreported to be lower than that of other known substrates [47],implying that Cys–SS–Cys is a preferred substrate.

As noted immediately above, alkyl hydropersulfide biosynthesisdoes not require initial H2S generation. Since hydropersulfides canbe degraded in biological systems to generate H2S (for example, viathe reverse of Reaction (2), it is proposed that H2S can serve as a“marker” for hydropersulfides [40]. Indeed, it has been furtherconsidered that hydropersulfides can be the actual major effectorspecies in biological signaling [40,45].

If hydropersulfides are biologically important effector speciesand if a major source of biological hydropersulfide (i.e., Cys–SSH) isfrom Cys–SS–Cys via CSE or CBS catalytic activity, then the levelsand regulation of Cys–SS–Cys may be an important issue regardingRSSH formation. Currently, it is known that the predominant formof cysteine in extracellular space/fluids is the oxidized Cys–SS–Cysform (for example, [48]), although both reduced and oxidizedforms exist. Interestingly, cysteine (Cys–SH) and Cys–SS–Cys aretaken up by cells via distinct transporters. Cys–SH is taken up by aNaþ-dependent neutral amino acid transporter. On the otherhand, Cys–SS–Cys is taken up by a Naþ-independent transporter,designated x–c , that exchanges Cys–SS–Cys and glutamate [49,50].The x–c transport system is composed of two disulfide-linkedsubunits, xCT, which mediates the transport, and 4F2hc, which isrequired for cell surface expression of the complex (reviewed by[51]). Initial studies focused on the import of cystine into cells dueto its presumed importance in GSH production; however, morerecently, attention has focused more on the efflux of glutamatedue to its potential role in excitatory signaling in the centralnervous system. Interestingly, mice deficient in xCT are healthyand fertile with no signs of neurologic defects reported; however,analysis of amino acids in the plasma revealed approximatelytwice the concentration of cystine [52]. Additionally, fibroblastsand macrophages derived from xCT-deficient mice failed tosurvive in culture without a reducing agent in the media such as2-mercaptoethanol [52,53]. Deficient macrophages also showedimpaired survival following activation by lipopolysaccharide (LPS),presumably due to increased sensitivity to oxidative stress causedby the respiratory burst. Importantly, this transporter is inducedby a variety of cellular stresses including oxidative stress (i.e.,H2O2) [54]. Uptake of Cys–SS–Cys into cells may be expected tolead to higher Cys–SH levels due to rapid intracellular reduction.However, it is reported that Cys–SS–Cys concentrations can berelatively high (approximately 30 μM in HT29 colon epithelialcells) and that the Cys–SH/Cys–SS–Cys ratio is only around 4(130 μM Cys–SH/30 μM Cys–SS–Cys) compared to a GSH/GSSGratio in the same cells of over 150 [55]. (Significantly, more recentstudies indicate that the GSH/GSSG ratio can be much higher thangenerally reported [56]). It was also observed that the Cys–SH/Cys–SS–Cys and GSH/GSSG redox systems are independent of eachother and not metabolically coupled (that is, changes in GSH/GSSGratios are not mirrored by a change in Cys-SH/Cys–SS–Cys ratios)[55,57]. Moreover, it is also reported that the Cys–SH/Cys–SS–Cysredox couple is independent of thioredoxin (Trx1) [55]. These maybe important considerations in evaluating the role of Cys–SS–Cysin thiol redox physiology. Since it appears that significant intra-cellular concentrations of Cys–SS–Cys are maintained in a manner

that does not depend on the redox status of GSH/GSSG or Trx1 thisthen leaves the question of what is important in regulating Cys–SS–Cys levels (or the Cys–SH/Cys–SS–Cys ratio)? That is, what isprimarily responsible for converting Cys–SS–Cys to Cys–SH in acell? Currently, the primary mechanism by which Cys–SS–Cys isreduced to Cys–SH in cells is not established. Reduction of Cys–SS–Cys by GSH, glutaredoxin, or thioredoxin, although possible, islikely to be much too slow to be primary reduction pathways [55].Thus, if Cys–SS–Cys levels are found to be important/regulatory forthe generation of Cys–SSH, then this question will need to beanswered.

As discussed above, alkyl hydropersulfides, whether from Cys–SS–Cys metabolism or the reaction of H2S with disulfides, arelikely to be biologically relevant. This idea is supported by a recentreport indicating significant levels of Cys–SSH and glutathionepersulfide (GSSH) along with protein–persulfides in human andmouse tissues and cells in culture [40]. Of special note is theextremely high levels of GSSH (as high as 150 μM in mouse brain)and Cys–SSH (1–4 μM in mouse tissues) found in mammaliansystems. Thus, it is useful to carefully consider the chemistry andbiochemistry of persulfides as possible biological effector species. Atthe cellular level, H2S is rapidly consumed in mitochondria by SQR,which results in persulfide intermediates [58]. The Km for this processis 100 nM while mitochondrial consumption by another persulfide(i.e., GSSH)-metabolizing enzyme, ETHE1 (also known as persulfidedioxygenase), has a higher Km [59], thus predicting a biological“equilibrium” between H2S and GSS� to be in favor of persulfides.Persulfide intermediates can also be produced by 3-mercaptopyruvatesulfur transferase (3-MST) from cysteine/ketoglutarate (for example,[60]), which is higher in the brain and may account for the finding ofhigher persulfide in this tissue bed [40]. Thus, it appears that thepresence of persulfides can be maintained by a balance of metabolicprocesses that seems to favor their formation. The presence ofoxidants (i.e., H2O2) and Cys-SS-Cys and the recycling of H2S suggestthat persulfides may be the predominant species with H2S serving asa unique dosimeter for these species.

Alkyl hydropersulfide chemistry: Nucleophilic and electrophilicproperties

The biological chemistry of RSSH has not been examined exten-sively (at least compared to thiols). Perhaps the lack of description ofRSSH chemical biology is at least partially due to the fact that thegeneral biological relevance of these species has only recently beenreported (although they have been known to be biologically relevantin specific systems for some time; vide infra). Also, RSSH possess aninherent instability that makes them somewhat difficult to study—alkyl hydropersulfides will spontaneously decompose in a pH-dependent manner to give numerous products including the corre-sponding thiol, disulfide, sulfide, and elemental sulfur (S0 whichoften seen as insoluble S8) (Reaction (3)) (for example, [61]).

RSSH-RSHþS0þRSSRþH2S(not balanced) (3)

Although the details of the mechanism of this degradation are notestablished, this inherent instability is thought to prohibit thelong-term storage of alkyl hydropersulfides. Thus, examination of

NH3

SOOC S

NH3

COOCSE/CBS

NH3

SOOC SH O

COO+ NH4++ H2O

Cys-SS-Cys Cys-SSH Pyruvate (CSE)

HOCOO

NH3

Serine (CBS)

Fig. 1. Generation of Cys–SSH from Cys–SS–Cys.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–9486

RSSH chemistry/biochemistry is typically performed via in situ gen-eration either chemically (for example, [43,62]) or enzymatically [40]).

It has been proposed that alkyl hydropersulfides can be a“hyperactivated” form of a thiol [10,43,63]. That is, some of thebiologically relevant chemistries associated with thiol functioncould be accentuated when it is converted to a hydropersulfide.For example, thiol conversion to a hydropersulfide can increase thenucleophilicity of the reactive sulfur atom [40], make the sulfur abetter reductant [64], and, although not yet confirmed, likely formsa better metal ligand (most probably with “soft” metals). It is worthnoting, however, that these statements are primarily based onspecies “free” in solution and/or for thiols whose pKa is 47 (atpH 7). Protein thiols can be extremely reactive (as nucleophiles,reductants, metal ligands) due to the protein environment. Alkylhydropersulfides are more acidic than the corresponding thiol by 1–2 pKa units, indicating that a greater proportion of the anionicspecies (RSS�) will be present when thiols are converted to thecorresponding hydropersulfide. Although it can be envisioned thathydropersulfide formation on a protein active-site cysteine can leadto increased protein activity (due to, for example, increased nucleo-philicity for thiols with pKa 47 at pH 7), this is not firmlyestablished. Mustafa and coworkers [63] reported that hydroper-sulfide formation, via reaction of H2S with the enzyme glyceralde-hyde 3-phosphate dehydrogenase (GAPDH), led to an increase inenzyme activity as evidenced by an increase in Vmax (but no changein the Km). It needs to be noted, however, that an increase in Vmax

with no change in Km does not necessarily reflect the presence of akinetically distinct enzyme but instead may be more consistentwith the idea that H2S simply reduced oxidized/inactive enzymeleading to an increase in the amount of total active enzyme (thus anincrease in Vmax and no change in Km). Consistent with this idea isthe finding that hydropersulfide formation in a cysteine proteaseleads to an inhibited enzyme but further treatment with high levelsof H2S eventually leads to the fully reduced, active enzyme [43].Regardless, the idea of “hyperactivated” activity in proteins remainsto be established (at least with regard to the “normal” or estab-lished function of proteins). Indeed, currently most studies indicatethat protein persulfide formation leads to an inhibition of thiolprotein activity (for example, [65]).

The fact that alkyl hydropersulfides could be trapped by electro-philes and characterized indicates that they have nucleophiliccharacter (for example, [43,62]). The fundamental reactivity of ahydropersulfide would be expected to be a function of its protona-tion state. As the anion (RSS�), the terminal, negatively chargedsulfur atom should be nucleophilic due to the negative charge andan α-effect (increase in reactivity due to adjacent electron pairs, forexample, [66]). The nucleophilicity of a hydropersulfide (or persul-fide anion) versus the corresponding thiol was clearly demon-strated recently when it was shown that significant reaction withH2O2 was only seen under conditions where GSSH was formed, notGSH or HS� [40]. Also, it was reported that GSSH reacts with thebiological electrophile 8-nitro-cyclic guanosine monophosphate (8-NO2-cGMP) to displace the nitro group, eventually forming the 8-SH-cGMP [40]. Under the conditions of this study, other thiols,including H2S did not react with 8-NO2-cGMP, consistent with theidea that free alkyl hydropersulfides are significantly more nucleo-philic than free thiols. The observed increase in nucleophilicity withpersulfides compared to the corresponding thiols for the abovenoted studies may be due simply to the fact that a larger percentageof the persulfide anion is present compared to the thiolate anionunder the experimental conditions. Whether persulfide anions areinherently more nucleophilic than the corresponding thiolateanions (possibly due to an α-effect) remains to be determined.

The protonated alkyl persulfide species (RSSH) would not beexpected to be as nucleophilic as the persulfide anion species.However, RSSH would be expected to have electrophilic character.

That is, nucleophilic attack on either of the sulfur atoms is possible.The electrophilic character of RSSH has been proposed in thereaction of cyanide ion (CN�) with RSSH to give the correspondingthiol and thiocyanate (�SCN) (Reaction (4) (for example, [67,68]).

RSSHþCN�-RSHþ�SCN (4)

Importantly, the reaction of CN� with the RSSH species to givethiocyanate (sometimes referred to as “cyanolysis”) has been usedas evidence for the presence of a hydropersulfide in proteins (forexample, [69–71]) (more recently, a unique biotin switch assay wasdeveloped by Zhang et al. [72], which was successfully applied forthe detection of protein-bound cysteine persulfides in cells [40]).The mechanism of thiocyanate formation from the reaction of CN�

and hydropersulfide is intriguing and potentially important since itrepresents a fundamental reaction of hydropersulfides. This reac-tion can be envisioned to occur in several ways (Fig. 2).

Direct attack at the sulfane sulfur by CN� (Fig. 2a) would leaddirectly to the established thiocyanate and thiol products. (Note:sulfane sulfur is defined as a sulfur-bonded sulfur atom formallywith six electrons [73] and in this case the terminal sulfur atom.)This mechanism would imply greater electrophilicity associatedwith the sulfane sulfur atom. Alternatively, attack of CN� canoccur reversibly at either sulfur atom eventually leading to thethermodynamically most stable products, which in this casewould be �SCN and the corresponding thiol (Fig. 2b). Finally,tautomerization of a persulfide to give a thiosulfoxide (RS(S)H)would generate a reactive “singlet sulfur” sulfane species whichwould be expected to be very electrophilic and reactive towardnucleophiles such as CN� (Fig. 2c) [73]. The electrophilic characterof sulfane sulfur species was recently used in the development ofreaction-based fluorescent probes for sulfane sulfur [74].

The mechanisms shown in Fig. 2 are perhaps problematic whenconsidered in a biological context. For example, when comparingRS� versus HS� as possible leaving groups, it may be predictedthat HS� will be superior to RS� in this regard (since the pKa of atypical, free RSH is 47 and the pKa of H2S is approximately 7,indicating that the HS� anion is more stable than the RS� anionand therefore a better leaving group). Discounting other factors,this predicts a reaction at the internal (rather than the terminal)sulfur atom at pH 7. Of course, in proteins, the pKa values of theconjugate acids of the leaving RS� groups may vary significantly,

Fig. 2. Possible mechanisms of �SCN formation from reaction of CN� with apersulfide.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–94 87

making it hard to predict which would be a better leaving group,RS� (or protein–S�) or HS� . For the analogous reaction of CN�

with unsymmetrical disulfides (RSSR0), the products are primarilybased on the formation of the most stable thiolate anion [75],which would predict predominant nucleophilic attack at theinternal sulfur atom for RSSH. It can be argued that the terminalsulfur atom is less sterically hindered, therefore favoring reactionat that site. Indeed, sterics have been reported to play a role in thisreaction [76]. Although the reaction could be under thermody-namic control (as would be the case for reaction b in Fig. 2)predicting that the product would be �SCN and the thiol, therelevance of this mechanism to a biological system may bequestionable since many other competing reactions associatedwith the products are possible, and this may preclude setting upequilibrium processes. Finally, the tautomerization of a hydroper-sulfide to a thiolsulfoxide has been examined theoretically and thefavorability of this process appears to depend significantly on themolecule [77–80]. Generally, using HSSH as a model hydropersul-fide, it appears that the thiosulfoxide tautomer (H2SS) is higher inenergy than the corresponding hydropersulfide (HSSH) by approxi-mately 20–40 kcal/mol. Interestingly however, for disulfide difluor-ide (FSSF) the thiosulfoxide (F2SS) is reported to be more stable thanthe disulfide by a few kcal/mol, indicating that the relative stabilityof these two isomers can be significantly altered by its elementalcomposition (and possibly a protein environment). Regardless, it isclear that RSSH possesses an electrophilic sulfur atom that iscapable of reacting readily with nucleophiles (such as CN�).

One-electron redox chemistry of persulfides

Thiyl radicals (RS � ) are common in biological systems, mostlypresent in oxidizing enzymes such as ribonucleotide reductase (forexample, [81]). The reduction potentials for thiyl radicals have beenreported to be slightly less than 1 V (i.e., for the GS � ,Hþ/GSH couple,E10 ¼0.92 V, pH 7.4 [82]), indicating that thiyl radical species can bereasonable one-electron oxidants (and that thiols/thiolates are onlymoderate one-electron reductants). Although the reduction potentialfor RSS � is not reported, it has been shown that RSSH/RSS� is morereducing than RSH/RS� . The S–H bond of RSSH is approximately21–22 kcal/mol weaker than the S–H bond of RSH [64] (approxi-mately 70 kcal/mol for RSS–H versus 92 kcal/mol for RS–H [83]). Thereason for the decrease in bond strength for RSSH can be attributedto the increased stability of the perthiyl radical (RSS � ) compared tothe thiyl radical (RS � ). The odd electron of RSS � is resonance-stabilized by the adjacent sulfur atom, an affect unavailable to theodd electron of a thiyl radical (Fig. 3). The resonance effect respon-sible for the stability associated with the perthiyl radical is akin to theeffect that stabilizes, for example, nitroxide radicals (R2NO·).

It is important to note that the perthiyl radical has an absorbanceat 374 nm (ε374¼approx. 1700 M�1 cm�1) that is distinct from thosetypically observed for thiyl radicals, which allows facile detection ofperthiyl intermediates in reactions [64]. The more stable perthiylradical is responsible for the increased propensity for H-atomdonation and reductive electron transfer to oxidants by hydroper-sulfides compared to the analogous thiol species [64], thus allowinghydropersulfides to be superior antioxidants. That is, reaction ofhydropersulfides with one-electron oxidants (such as an organicradical species, R·) is more favorable than the analogous reactionwith thiols (Reaction (5)) and due to the relative stability of theperthiyl radical, further oxidation chemistry via perthiyl-mediatedoxidation is diminished.

RSSH/RSS�þR � (oxidant)-RSS � þRH/R� (5)

As expected, single electron reduction of metalloproteins is alsosignificantly more prevalent with persuflides than with thiols. For

example, hydropersulfides can interact with ferric heme proteinsresulting in reduction to the ferrous species (i.e., [43,64,84]).Although H2S and thiols can also serve as reducing agents, itappears that hydropersulfides are far superior in this regard. Analkyl hydropersulfide was also reported to inhibit the activity ofthe heme protein cytochrome P450 by altering the heme moietyby an as yet undetermined fashion [85].

As shown above, one-electron oxidation of hydropersulfides/per-sulfide anion generates the perthiyl radical and, generally, perthiylradicals are presumed to be less oxidizing than the corresponding thiylradicals. However, perthiyls will react with O2 (k¼5�106 M�1 s�1) togive initially a perthioperoxyl radical (RSSOO � ) and ultimately tosulfate (SO2–

4 ) [86]. It is presumed that sulfate formation results fromrearrangement of RSSOO � to give the sulfonyl-type radical (RSS(O)O � .In a purely chemical system, SO2–

4 generation is presumed to resultfrom bimolecular dimerization of RSS(O)O � species followed byfurther degradation. The bimolecular nature of this process clearlylimits its relevance in biological systems. Perthiyl radicals will alsoreact with biological reductants such as ascorbate (RSS � þAH�-RSSHþA � � , k¼4.1�106 M�1 s�1), although expectedly at a decre-ased rate compared to the more reactive thiyl radicals (RS � þAH�-RSSHþA � � , k¼4.9�108 M�1 s�1) [86]. Although speculative at thistime, it is alsoworthwhile to note that akin to thiyl radical species [87],perthiyl radicals may also react rapidly with thiolates present in abiological milieu to give a trisulfide radical anion (RSSSR � �). In thecase of disulfide radical anions (RSSR � � , formed from the reaction ofRS � with RS�), these species are very good reductants, capable ofreducing O2 to superoxide (O�

2 ). Whether the equivalent chemistrycan occur with putative RSSSR � � species remains to be determined.

Two-electron redox chemistry of hydropersulfides

Oxidation of alkyl hydropersulfides with 2e� oxidants such ashydrogen peroxide (H2O2) occurs readily. Indeed, RSSH species arereported to react with H2O2 significantly faster than thiols andoverexpression of the persulfide-synthesizing enzyme CSE affordsprotection of cells from H2O2 toxicity [40]. Although not established,it appears likely that reaction of alkyl hydropersulfides/persulfideanion with H2O2 will give an alkyl perthiosulfenic acid (not to beconfused with a persulfenic acid, RSOOH, vide supra) (Reaction (6)).

RSSHþH2O2-RSSOHþH2O (6)

Furthermore, analogous to thiol chemistry where sulfenic acidsreact with thiols to give H2O and disulfides (for example, [88]),perthiosulfenic acids are likely unstable with respect to reactionwith excess persulfides or thiols to give a disubstituted tetrasul-fide/trisulfide species (Reactions (7) and (8).

RSSOHþR0SH-RSSSR0 þH2O (7)

RSSOHþR0SSH-RSSSSR0 þH2O (8)

Interestingly, significant levels of tri-, tetra- and higher polysulfideshave been detected in mammalian tissues and plasma, alluding tothe possible and significant relevance of oxidative polysulfidegeneration from initial hydropersulfide biosynthesis [40].

Fig. 4 schematically depicts all of the chemistry discussed forhydropersulfides. To be sure, some of this chemistry is speculativeor currently unknown and, for certain aspects, based on extendingknown thiol chemistry. Regardless, this scheme can serve as astarting point and basis of further investigation and, at the veryleast, depicts the diverse chemistry of hydropersulfides.

R S S R S S

Fig. 3. Resonance stability of the perthiyl radical.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–9488

Chemistry of dialkylpolysulfides and alkyl hydropolysulfides

The formation of H2S in biological systems allows for theformation of alkyl hydropersulfides/persulfide anions, which in turnallows for the formation of dialkylpolysulfides (R–Sn–R, n42)(Reactions (2), (6)–(8)) (although as noted above, alkyl hydropersul-fides can be made via H2S-independent mechanisms, which mayhave greater biological importance). Moreover, it appears thatdialkylpolysulfides are biologically prevalent [40]. Therefore, it isworth considering the chemistry of dialkylpolysulfides that may beresponsible for their biological fate/function (if any). Previous interestin dialkylpolysulfides, at least from a biological perspective, is due tothe fact that they are major components of garlic and other foodswith presumed health benefits (for example, [89]). Interestingly,dialkylpolysulfides have also been implicated as toxic species capableof eliciting hemolytic anemia in certain animals (for example, [90]).Regardless, the chemistry responsible for either the presumedbeneficial or the deleterious effects of dialkylpolysulfides is notestablished.

Like hydropersulfides, dialkylpolysulfides are capable of actingas electrophiles. Reaction of a dialkylpolysulfide (e.g., glutathionetrisulfide, GSSSG) with a nucleophilic thiol results in a substitutionreaction of the type shown below for a dialkyltrisulfide (Reactions(9) and (10)).

RSSSRþR0SH-RSSHþR0SSR (9)

RSSSRþR0SH-RSHþR0SSSR (10)

The factors that govern the position of the nucleophilic attack,in the case of a dialkyltrisulfide, have not to our knowledge beenestablished. It is likely that steric effects would favor nucleophilicattack on the sulfur atom(s) furthest away from the R group andare bound to two sulfurs with large electronegativities (as inReaction (10)). However, since RSSH is more acidic than RSH(making RSS� a better leaving group than RS�) it may be expectedthat this will favor nucleophilic attack on a sulfur atom that wouldallow expulsion of an alkyl hydropersulfide/persulfide anion (oralkyl hydrogen polysulfide, RS–Sn–H as in Reaction (9). Signifi-cantly, Massey and coworkers [84] reported that Reaction (9) wasresponsible for an interesting effect whereby GSSG was able toenhance the rate of reduction of ferric cytochrome c by GSH. Thisenigmatic finding initially indicated that an oxidant, GSSG, wasable to assist the reductant, GSH, in reducing ferric cytochrome c.Massey was able to explain this phenomenon by showing thatcommercial GSSG contained significant amounts of the polysulfideGSSSG, which can react with GSH to give GSSH (via Reaction (9)).As discussed previously, hydropersulfides/persulfide anions are

superior one-electron reductants compared to thiols, thereforeallowing more facile ferric cytochrome c reduction. Reduction offerric cytochrome c by an alkyl hydropersulfide/persulfide anionwould generate the corresponding alkyl perthiyl radical (RSS � )(Reaction (11)).

RSS�/RSSHþFeIII�cyt c-FeII�cyt cþRSS � þ(Hþ) (11)

In a simple chemical system, RSS � may have the opportunity todimerize to give, in this case, the dialkyltetrasuflide (RSSSSR)(Reaction (12)) (as noted above, reactionwith O2 is possible as well).

RSS � þRSS �-RSSSSR (12)

Further reaction of RSH with the dialkyltetrasulfide would thengenerate, among other possible products, the dialkyltrisulfide andan alkyl hydropersulfide (Reaction (13)).

RSSSSRþR0SH-R0SSSRþRSSH (13)

Therefore, Reactions (12) and (13) allow GSSSG to be catalyticin facilitating the overall reduction of ferric cytochrome c by GSH(via hydropersulfide formation). Of course, in a biological system,many other processes/reactions may intercept reactive intermedi-ates like alkyl perthiyl radicals or even hydropersulfides. However,this example is important in illustrating the redox exchangechemistry that exists for dialkylpolysulfides.

The above discussion indicates that dialkylpolysulfides can act aselectrophiles. Indeed, due to the enhanced ability for hydropoly-sulfides/polysulfide anions (such as an alkyl hydropersulfide) to actas leaving groups in substitution reactions, dialkylpolysulfides areassuredly more electrophilic than the corresponding disulfides.

The presence of extended dialkylpolysulfides (i.e., dialkyltetra-sulfides, dialkylpentasulfides, etc.) allows for the formation of alkylhydropolysulfides with more than two sulfur atoms. For example,reaction of a dialkyltetrasulfide with a thiol can give, among otherpossible products, an alkyl hydrotrisuflide (Reaction (14)).

RSSSSRþR0SH-RSSSHþR0SSR (14)

The factors that make hydropersulfides more reactive than thecorresponding thiols (as nucleophiles, acids, reductants; vide supra)should also make alkyl hydrotrisulfides (RSSSH) more reactive thanalkyl hydropersulfides. For example, it is known that hydropersulfidesare more acidic than the corresponding thiol (vide supra). It is alsoreported that for a series of dihydropolysulfides (HSnH, n¼1–5),increasing the number of sulfur atoms leads to decreasing pKa (H2SpKa¼7.0, H2S2 pKa¼5.0, H2S3 pKa¼4.2, H2S4 pKa¼3.8, H2S5 pKa¼3.5)[91]. It seems likely that increasing the number of consecutive sulfur

R S S H R S S + H+

pKa approx. 7very nucleophilicelectrophilic

good one-electron reductant

H-atomdonor?

Nuc

Nuc-S + RSH

E

RSSE

Mn+

R-S-S· + M(n-1)+

R'-S-S-R' or R'-S-OH

R-S-S-S-R' + R'SH or H2O

RS

H

S

RS

H

S Nuc

RS

H

S ?

R·

RSS· + R-H

R·,H+

Fig. 4. Possible/putative biologically relevant reactions of persulfides.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–94 89

atoms will also lead to increased nucleophilicity and one-electronreducing capability as well.

Comparison of persulfide chemistry with peroxide chemistry

Clearly, much is known regarding the chemistry and biologicalactivity of peroxides (ROOH, ROOR, HOOH) (for example, [see 92]).Based on their structural similarity there may be the tendency toextrapolate known peroxide (e.g., ROOH) chemistry to that ofpersulfide chemistry. To some extent, this seems reasonable. Forexample, H2O2 acts as a nucleophilic oxidant under basic condi-tions via HOO� chemistry and an electrophilic oxidant underacidic conditions via HOOHþ

2 (or the equivalent) chemistry (Fig. 5).As noted above, the same scenario is proposed for hydroper-

sulfides (i.e., as the anion it is a nucleophilic oxidant and underacidic conditions the neutral (or protonated) form is an electro-philic oxidant, Fig. 4). It is worth noting, however, that the pKa ofRSSH is in the range of 6–8 (in the physiological pH range)whereas the pKa of H2O2 is 11.8 and far removed from physiolo-gical pH. Thus, the anionic chemistry of hydropersulfides is clearlymore accessible under biological conditions compared to H2O2

(and other alkyl hydroperoxides, ROOH). Although there are somepotentially important chemical similarities between persulfidesand peroxides, there are also some important differences as well.These chemical differences are best illustrated by comparing someof the basic physical constants associated with these two func-tional groups. The O–O bond dissociation energy (BDE) of aperoxide is dependent on the substitution and in the range of36–51 kcal/mol (the BDE for HO–OH is 51 kcal/mol and the BDE forCH3O–OCH3 is 36 kcal/mol). On the other hand, the BDE for an S–Sbond in a persulfide is significantly higher. The BDE for HS–SH is66 kcal/mol and for CH3S–SCH3 the BDE is 74 kcal/mol [83]. Thelow BDE for peroxides (at least compared to persulfides) can beexplained by the weakening of the O–O bond due to repulsionbetween the adjacent lone pairs of electrons on the adjacentoxygen atoms. Since the S–S bond is significantly longer comparedto the O–O bond of a peroxide (2.05 Å versus 1.48 Å, respectively)and the electron pairs are in larger, more diffuse orbitals, theelectron-pair repulsion is lessened in the case of the persulfides.Significantly, increasing the number of sulfur atoms (to makepolysulfides) decreases the S–S BDE. For example, the S–S BDEsfor the series HS–SH, HS–SSH, and HSS–SSH are 66, 50, and33.6 kcal/mol, respectively [83]. The rationale for this is the sameas that given previously for explaining the stability of RSS �compared to RS � (Fig. 3). Thus, any reaction whose product is aperthiyl radical will be more favorable than a correspondingreaction that generates a simple thiyl radical product. By analogy,polyoxides should follow the same trends. However, the stability

of the polyoxides (i.e., H2O3, etc.) is significantly lower with onlyscant and controversial reports of their biological relevance (forexample, [93,94]). To illustrate this, consider that the HO–OH, HO–OOH, and HOO–OOH BDEs are reported to be only 51, 25, and 7–12 kcal/mol, respectively [95]. Thus, it appears that polysulfidespecies are biologically accessible whereas polyoxides, if formed,would be considerably less stable.

The reactivities of the perthiyl radical and the peroxyl radical arealso quite different. For example, the S–H BDE for HSS–H isapproximately 70 kcal/mol, whereas the O–H BDE for HOO–H is90 kcal/mol [83]. Thus, HOO � (which is simply protonated super-oxide, O�

2 þHþ ) is a significantly better one-electron oxidant com-pared to HSS � (or RSS � ). This also implies that RSSH can act as anantioxidant (as noted previously) via H-atom donation, whereasROOH is never considered an antioxidant and has always beenviewed as an oxidant. That is, reaction of RSSH with a strong one-electron oxidant will quench the oxidant and generate a fairly stableradical, RSS � (RSSHþR �-RSS � þR–H), which is a poor oxidant. Onthe other hand, reaction of a strong one-electron oxidant with ROOHin the equivalent manner will give another strong oxidant ROO � .

One of the most studied reactions of peroxides is the Fentonreaction whereby a peroxide (such as H2O2) is reduced by a metalto generate the oxidized metal and the very strong one-electronoxidant, hydroxyl radical (HO � ) (Reaction (15)) [92].

H2O2þMn-HO � þMnþ1þHO� (15)

(Mn¼Fe2þ , Cu1þ; Mnþ1¼Fe3þ , Cu2þ)

From a pathophysiological/toxicological perspective, the Fentonreaction may be important since it represents a counterintuitiveprocess whereby reductants (e.g., reduced metals and biologicalspecies capable of reducing metals) are able to create a potentoxidant, HO � . The potential for the H2O2,Hþ/HO � ,H2O (pH 7, vsNHE) couple is 0.320 V [96]. Presumably, the relative favorabilityof this process is due in part to the breaking of a weak O–O bondand the generation of the extremely stable H2O molecule. Thereduction potential for HSSH has not, to our knowledge, beenreported so a direct comparison to H2O2 is not yet possible.However, the reduction potential for the RSSR/RSSR � � couple isreported to be approximately –1.4 V, or extremely unfavorable[97,98]. It should be noted that the product of the one-electronreduction of RSSR is not chemically analogous to that shown forthe Fenton reaction (HO � þHO�). The product of RSSR reduction istypically viewed as being the disulfide radical anion (RSSR � �) asopposed to the thiyl radical and thiolate/thiol species (RS � þRS�).It has been shown that the equilibrium constant for the reaction ofthiyl radical and thiolate (Reaction (16)) is in the range of 33–2000, depending on the R group, indicating a predominance of theradical anion dimer [99].

RS � þRS�⇌RSSR � � (16)

RSSR � � is a strong one-electron reductant as the extremelynegative reduction potential for RSSR indicates (as opposed to thestrong oxidant, HO � , formed form the Fenton reaction). Thus, anoxidizing “Fenton-like” reaction associated with persulfides appearsunlikely. Finally, the two-electron reduction potential for H2O2 is1.32 V [92] and for ROOH approximately 1.28 V (both pH 7) [100],both of which are positive and relatively favorable. Thus, reactionswhereby substrates are oxidized by two electrons can occur readilywith peroxides (although there can be kinetic restrictions to thischemistry). On the other hand, the two-electron reduction potentialfor RSSR is only approximately 0.08 V [98] (note: the two-electronreduction potential for H2S2 is not reported), indicating that directtwo-electron oxidations via persulfides are not as favorable as it iswith peroxides. Based on this brief discussion, hopefully it isevident that there are similarities between persulfides/polysuflides

H O O H+H+

-H+H O O H

H+H+

-H+H O O

Nuc

Nuc-OH + H2O

ElectrophileNucleophile

H

O

H

OO

+ HO

Fig. 5. H2O2-mediated oxidations under basic and acidic conditions.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–9490

and peroxides/polyoxides but that the biological relevance orimplications of these chemistries can be distinct and even opposite.Thus, care must be taken when extrapolating peroxide chemistry toexplain the biological mechanisms of persulfides.

Possible biological implications of persulfide and polysulfidegeneration

The chemical properties of persulfides and polysulfides dis-cussed above indicate enhanced and distinct reactivity comparedto thiols. Moreover, it is now clear that relatively high levels ofthese species are present in mammalian tissues and plasma. Theoverriding question that remains is: What are the functions/rolesof per- and polysulfides present in biology? That is, why hasNature chosen to generate these species? Of course, it is far tooearly to present any definitive answers to this question. However,it is at least worthwhile to speculate on the biological functionsand roles of these species since something is known about theirchemical properties and it is reasonable to contend that theirfunctions are based on their unique or novel chemistries.

As noted earlier, it has been proposed that hydropersulfidegeneration could represent a “hyperactivation” of thiol reactivitywhere aspects of biological thiol chemical reactivity are enhanced.On the other hand, as noted above, hydropersulfide formation canalso lead to an inhibition of thiol protein activity, as has beenreported numerous times already. To date, protein hyperactivationhas not been unequivocally demonstrated (vide supra). Based on therecent findings that numerous proteins contain persulfides [6,40], itis reasonable to suspect that formation of these species is a generalphenomenon that alters the activity/function of numerous systemswithin a cell (likely as the result of a general cellular stress orsignal). However, it should be noted that some methods used foridentification of protein persulfides are questionable (see detaileddiscussions in [62,72]). It is necessary to develop more sensitive,selective, and “easy to use” methods for persulfide detection. Withrespect to hydropersulfides (which are the precursor to all poly-sulfides and higher hydropolysulfides) it is worthwhile at this pointto “step back” and examine the likely biological conditions con-ducive to its biosynthesis, the consequences of its formation, and itspossible physiological role. First and foremost, it needs to bestressed that a hydropersulfide is OXIDIZED with respect to a thiol.That is, the formation of an –SSH group requires an oxidation of an–SH function. This is analogous to the relationship between a thiol,RSH, and a corresponding disulfide, RSSR. Thus, generation of ahydropersulfide requires an initial oxidizing event. In the case ofbiological hydropersulfide formation, the oxidizing event (or theoxidized species that must be present) can be the formation of Cys–SS–Cys from Cys–SH. As noted above, CSE-mediated conversion ofCys–SS–Cys to Cys–SSH is a major pathway of biological persulfide/polysulfide generation. The levels of intracellular Cys–SS–Cys havebeen reported to be in the range of 30 μM (in colon epithelial cells)[55]. Significantly, the Km of Cys–SS–Cys for CSE is also approxi-mately 30 μM [47], indicating that Cys–SS–Cys levels may beregulatory for Cys–SSH generation. As described previously, Cys–SS–Cys levels are regulated independently from the GSH/GSSGsystem, with Cys-SH/Cys–SS–Cys ratios being significantly lowerthan GSH/GSSG ratios. It is tempting to speculate that maintaininghigher levels of Cys–SS–Cys may be due to an important biologicalrequirement to maintain Cys–SSH generation capabilities (withsubsequent formation of other persulfide/polysulfide species). Ifthe presence of the oxidized thiol Cys–SS–Cys is an importantregulatory factor for hydropersulfide formation, then factors thatpromote or cause an increase in Cys–SH oxidation to Cys–SS–Cyscan promote increased hydropersulfide formation (with subsequentincreases in GSSH, protein–SSH, etc.). These factors can be, of

course, oxidants such a peroxides or oxidizing nitrogen oxides (e.g., NO2, N2O3, etc.). As described above, hydropersulfides (and theirmore prevalent anions) are superior one- and two-electron reduc-tants compared to their corresponding thiols. What this indicates isthat oxidizing conditions (e.g., high H2O2 or high Cys–SS–Cys levels)can lead to the direct formation of oxidized thiol species that arepowerful reductants (e.g., Cys–SSH, GSSH, etc.). Moreover, normalcellular reducing conditions would lead to reduction back to thenormal thiol. That is, the direct biosynthesis of a potent reductant,Cys–SSH and derived species, can be the result of an oxidizingenvironment. This represents a novel and fascinating possibilitythat could be an important and immediate first step in protectingcells from the immediate ravages of oxidants. If indeed persulfide/polysulfide generation represents a protective strategy, the nextquestion would be: Why would persulfides/polysulfides be protec-tive? As shown by Everett and Wardman [64], hydropersulfides arepotent antioxidants due to their reducing capabilities. For example,GSSH could directly scavenge H2O2 [40] or rapidly quench one-electron oxidants (such as oxidizing radical species). It is worthpointing out a unique factor associated with the antioxidantpotential of hydropersulfides—oxidation of persulfides followed byreduction can generate back the original thiol species. As shown inFig. 6, persulfide formation in proteins could make them especiallyreactive with oxidants (in this case H2O2 will be used as aprototypical oxidant). Stepwise oxidation of a persulfide withH2O2 leads to perthiosulfenic, perthiosulfinic, and perthiosulfonicacids. All of these species would likely inhibit protein activity.However, under normal reducing conditions, all of these speciescan be reduced to give back the original thiol proteins. In the case ofthiol protein oxidation, for the most part, oxidation past the sulfenicacid state would irreversibly inhibit the thiol protein and regainingbiological activity would likely require de novo protein synthesis.Thus, the sacrificial sulfur associated with protein persulfide gen-eration can allow oxidation and protein thiol regeneration whennormal cellular redox conditions are reestablished.

Indeed, protection of cysteine phosphatase activity against irre-versible oxidation by H2O2 via presumed hydropersulfide formationhas been observed, although the mechanistic details of this protec-tion have not been discussed [65,101]. Significantly, the oxidation ofalkyl hydropersulfides and eventual reductive regeneration of thecorresponding thiol have further biological precedence. The sulfide-metabolizing enzyme ETHE1 has been reported to specifically oxidize

Cys-SSH, GSSH

[O]

PS

S

O

O

P S S

O

O

O

P-S PSS

(P = Protein)

PS-S-O

[H]P-S + SOx

n-

Protein Persulfide

Protein Thiol

PS

O

O

P S

O

O

O

P-S

(P = Protein)

PS-O[H]

P-S[O]

irreversibleinhibition

Fig. 6. Possible role of protein persulfide formation.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–94 91

hydropersulfides to the corresponding perthiosulfonic acid, whichthen is reduced to give sulfite (SO2–

3 ) and thiol [59,102].Thus, protection of cells, proteins, and biological macromole-

cules from oxidants via persulfide formation can be due to twofactors: (1) direct scavenging of oxidants, and (2) formation of asacrificial sulfur species in proteins. Scheme 1 depicts thesepossible roles of persulfide/persulfide formation in biologicalsystems. Due to the relative newness of this field, the proposedprotection afforded by biological persulfide generation currentlyhas only scant support [40] and the ideas presented herein willneed to be tested carefully. However, the idea that hydropersulfidegeneration can play a protective role makes chemical sense andseemingly can make physiological sense since it combines the“modified” thiol chemistry alluded to earlier with a physiologicalprotective function (that is not related to the actual proteinfunction). Regardless, there is little doubt that the general areaof investigation of the mechanism(s) of per- and polysulfidefunction (as well as H2S function) will be a topic of significantresearch interest for many years to come.

Acknowledgments

P.N. is grateful for financial support from FP7-PEOPLE-2010-RG(Marie Curie International Reintegration Grant PIRG08-GA-2010-277006) and The Hungarian National Science Foundation (OTKA;Grant K109843). The authors also acknowledge support from theNIH (HL106598 to J.M.F.; R01HL116571 to M.X.), NSF (CHE-1148641to J.M.F.), and ACS-PRF (52801-ND4 to D.J.T.). T.A., T.S., and Y.K.acknowledge support by Grants-in Aid for Scientific Research fromthe Ministry of Education, Culture, Sports, Sciences, and Technol-ogy (MEXT), Japan. P.N., J. M. F. and A. J. H. also acknowledge COSTaction BM1005 for providing networking opportunities.

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Cys-SH[O]

[H]Cys-SS-Cys

reduced oxidized

CSE/CBSCys-SSH

potent reductant

[O] = biological oxidants, stress[H] = reducing, normal conditions

GSSH Protein-SSH

scavenges oxidants

proteinprotectionfrom oxidants

GSH Protein-SH[H] [H]

H2S

Function (heme-proteins,reaction with oxidized thiols, etc.)

Scheme 1. Possible roles of persulfide and protein persulfide generation.

K. Ono et al. / Free Radical Biology and Medicine 77 (2014) 82–9492

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