Chemical Biology of H2S Signaling through Persulfidation

Abstract

Signaling by H₂S is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH). Persulfidation provides a framework for understanding the physiological and pharmacological effects of H₂S. Due to the inherent instability of persulfides, their chemistry is understudied. In this review, we discuss the biologically relevant chemistry of H₂S and the enzymatic routes for its production and oxidation. We cover the chemical biology of persulfides and the chemical probes for detecting them. We conclude by discussing the roles ascribed to protein persulfidation in cell signaling pathways.

Figure 1

Overview of different classes of H₂S donors. (A) Compounds based on the N-mercapto template (N-SH species) and the proposed mechanism for H₂S release. PG, protective group. (B) Perthiol-based compounds and proposed thiol-dependent mechanism of H₂S release. (C) Dithioperoxyanhydrides can also serve as H₂S donors upon reaction with thiols. (D) Arylthioamides release H₂S in the presence of thiols via an uncharacterized mechanism. (E) S-Aroylthiooximes release H₂S in the presence of aminothiols. (F) Chemical structures of Lawesson’s reagent and its derivative, GYY4137, the most widely used H₂S donor, and the proposed mechanism for H₂S release from GYY4137. (G) Phosphorothioate-based H₂S donors that release H₂S in a pH-dependent manner. (H) Another widely used class of molecules is 1,2-dithiole-3-thiones. They can be coupled to nonsteroid antiinflammatory drugs (NSAID) such as aspirin, ibuprofen, or naproxen, or to triphenylphsophonium group (AP39) which directs them to mitochondria. This class of molecules is believed to release H₂S via hydrolysis. (I) Example of photo cleavable gem-dithiol based H₂S donors, which undergo hydrolysis to release H₂S. (J) Thioamino acids release H₂S in reactions with bicarbonate. (K) COS, released by COS donors, forms H₂S in the presence of carbonic anhydrase. (L) Ammonium tetrathiomolibdate is shown to act as H₂S donor in vivo.

Figure 2

Organization and structure of human CBS. (A) CBS is a modular protein with regulatory domains at its N- and C-termini. The C-terminal domain comprises a tandem repeat of two CBS domains, CBS1 and CBS2. The structures of human CBS in the absence (PDB: 4L27) (B) and presence (PDB: 4PCU) (C) of AdoMet show that a large conformational rearrangement accompanies the transition from the basal to the activated state. The protomers are shown in blue and yellow, respectively, the heme (red) and PLP (yellow) are in sphere representation, and the blue arrows point to the intervening linker region between the catalytic core and the C-terminal domain.

Figure 3

Close up of the CBS structure. The interactions between the Cys52 heme ligand and Arg266 at one end of the α-helix and between Thr257 and Thr260 and the phosphate group of PLP at the other are shown. Asn149 hydrogen bonds with the C4 oxygen in PLP.

Figure 4

Structure of human CSE (PDB: 2NMP). Each of the four monomers is shown in a different color, and the three PLPs visible in the structure are in sphere representation.

Figure 5

Close-up of the active site structure of human CSE. (A). Interactions between PLP (yellow) and residues donated by the two subunits (in green and cyan) are highlighted (PDB: 2NMP). (B). Structure of PPG-inactivated CSE (PDB: 3COG). The coloring is the same is in panel A. PPG is covalently linked to Tyr114 and is shown in pink.

Figure 6

Heme-regulated switching in the transsulfuration pathway from cysteine (left) to H₂S (right) synthesis.

Figure 7

Structure and mechanism of human MST. (A) The N and C-terminal domains of human MST (PDB: 4JGT) are shown in blue and green, respectively, and the linker is in red. Pyruvate (cyan) and key active site residues including Cys248 in the Cys-SSH state are shown in stick representation. (B) Close up of the active site captured in a product complex with pyruvate (orange) and Cys-SSH (PDB: 4JGT). Two residues in the catalytic triad (D63 and H74) are donated by the N-terminal domain and are shown in blue. (C) Mechanism of the reaction between the 3-mercaptopyruvate substrate and the Cys248 thiolate.

Figure 8

Alternative models describing the organization of the mitochondrial sulfide oxidation pathway. (A) In this model, GSH is the sulfur acceptor from SQR and the product, GSSH, is utilized by either PDO or by rhodanese generating sulfite and thiosulfate, respectively. (B) In this model, sulfite is the sulfur acceptor from SQR and the product, thiosulfate, is utilized by rhodanese to generate GSSH, which is subsequently oxidized by PDO to sulfite. Sulfite is eventually oxidized to sulfate by sulfite oxidase. Q represents coenzyme Q.

Figure 9

Structure of SQR from A. ambivalens. (A). Structure of an SQR subunit (PDB: 3H8L) in which the covalently bound FAD is shown in stick representation. (B) Close-up of the active site showing FAD and a bridging trisulfide intermediate between Cys350 and Cys178.

Figure 10

Crystal structure of PDO. (A) Structure of human PDO (PDB: 4CHL) in which the protomers are shown in pink and blue, the iron ion as an orange sphere, and the coordinating histidines and aspartate in stick representation. Cys247 is oxidized as cysteine sulfinate and is also shown in stick representation. (B). Close up of the P. putida PDO (PDB: 4YSL) with bound GSH (green). The cysteine sulfur of GSH is proximal to the iron ion, which is coordinated by a 2His-1Asp facial triad.

Figure 11

Structure of bovine rhodanese (PDB: 1RHD). The N- and C-terminal domains are shown in green and blue with the intervening linker in pink. A Cys-SSH intermediate is stabilized at Cys247 in the active site, via hydrogen bonding interactions with neighboring residues.

Figure 12

Structure of dimeric chicken sulfite oxidase (PDB: 1SOX). The two subunits are shown in blue and magenta, respectively, and the heme (orange) and molybdopterin (MPT, cyan) cofactors are in sphere representation.

Figure 13

Biological reactivity of H₂S. H₂S can react directly with oxidants such as superoxide, HOCl, and ONOO⁻. It can also react with NO^• and S-nitrosothiols leading to the formation of other signaling species (right arrow). Metal centers in proteins can bind H₂S for delivery to specific targets, be reduced by H₂S, or catalyze sulfide oxidation chemistry (left arrow). H₂S is also involved in the modification of protein cysteine residues leading to persulfide (Cys-SSH) formation (central arrow).

Figure 14

Binding of H₂S to protein metal centers. (A) Structure of the α subunit of human hemoglobin showing HS⁻ bound at the entry/exit point of the so-called Phe path that leads to the distal face of the heme. A second sulfide is bound to the heme iron (PDB: 5UCU). (B) Close up of the heme in hemoglobin I from Lucina pectinata with HS⁻ coordinated to the iron ion (PDB: 1MOH). (C) Structure of hemoglobin-like protein C1 from Riftia pachyptila (PDB: 1YHU) with Zn²⁺ shown in blue and iron hemes in purple.

Figure 15

Signaling aspects of NO^•/H₂S cross-talk. (A) To form HNO and minimize side reactions, H₂S and NO₂^• have to be produced in proximity. HNO (and possibly HSNO) reacts with protein thiols and glutathione. (B) All three gases, NO^•, O₂, and H₂S tend to accumulate in membranes. NO^• and O₂ form N₂O₃, which readily reacts with H₂S to form HSNO. (C) HSNO formed in the reaction of protein S-nitrosothiols with H₂S can diffuse through the cell membrane and transfer the “NO⁺” group to another protein target.

Figure 16

Strategies for the preparation of protein persulfides. (A) Protein thiols react first with DTNB forming mixed disulfides that react with H₂S forming persulfides. Thionitrobenzoate is a good leaving group and its UV–visible absorbance can be used to estimate the yield of persulfides. (B) Protein sulfenic acids, when stable, can be used as precursors for persulfide preparation. (C) Protein thiols can be mixed with inorganic polysulfides or with a mixture of HOCl and H₂S. Besides persulfides, polythiolated products are also formed. (D) Protein thiols can react with 9-fluorenylmethyl disulfide. The products undergo alkaline hydrolysis forming persulfides.

Figure 17

Methodological approaches for the characterization of protein persulfides. (A) When pure, protein persulfides can be characterized by UV–visible, IR, and ¹H NMR spectroscopy. (B) Protein persulfides react with 1-fluoro-2,4-dinitrobenzene to form mixed disulfides. DTT releases 2,4-dinitrobenzenethiol, which absorbs at 408 nm under alkaline conditions. (C) Persulfides can be labeled with thiol blocking reagents such as iodoacetamide and analyzed by MS. (D) Protein persulfides can be tagged through different strategies that rely on either their electrophilic or their nucleophilic character.

Figure 18

Modified biotin switch assay for persulfide labeling. (A) MMTS was proposed to selectively block free thiols leaving persulfides unmodified and ready for reaction with a biotin-derivatized reactive disulfide (biotin-HPDP). (B) Mechanistic explanation for persulfide labeling with the biotin switch assay. MMTS reacts with persulfides more readily than with thiols forming a trisulfide product. This trisulfide is attacked by unreacted thiols leaving a free cysteine that can react with biotin-HPDP.

Figure 19

Persulfide detection by differential fluorescence tagging. Both thiols and persulfides are initially blocked with Cy5-maleimide. DTT treatment then removes the fluorescent tag from the persulfides. Proteins are separated by electrophoresis, and the loss of fluorescence caused by DTT is used as a measure of persulfidation.

Figure 20

Persulfide labeling with biotin-tagged alkylating reagents. (A) In the first step proteins are mixed with maleimide-biotin (or maleimide-PEG₂-biotin) to tag both thiols and persulfides. Proteins are trypsinized and the biotinylated peptides are bound to streptavidin beads. Persulfidated peptides attach to streptavidin beads via disulfide bonds. DTT treatment facilitates elution from the beads and subsequent MS analysis. (B) A possible caveat is that peptides connected by disulfide bonds and containing a thiol or persulfide could be released from the beads with DTT. However, the concentration of disulfide bonds in intracellular proteins is low and this not expected to be a quantitiatvely major drawback of the method.

Figure 21

Caveats of whole protein labeling with biotin-tagged alkylating reagents. Proteins containing several cysteine residues, of which only one is persulfidated, are labeled with biotin-maleimide (biotin-NEM) or biotin iodoacetamide (IAB). Since all labels have equal chances of binding to streptavidin beads, the elution with DTT will result in lower than expected yield because the protein will remain bound through the tagged thiols.

Figure 22

qPerS-SID (quantitative persulfide site identification) approach. Control cells and H₂S-treated cells are grown in heavy and light SILAC (stable isotope labeling with amino acids in cell culture) media, respectively. After cell lysis protein extracts are mixed 1:1 and exposed to iodoacetamide-PEG-biotin to labelthiols and persulfides. Proteins are then trypsinized and labeled peptides bound to streptavidinbeads. Since persulfidated peptides attach to streptavidin beads via disulfide bonds they are eluted with TCEP (tris(2-carboxyethyl)phosphine). Released peptides are subjected to LC-MS/MS identification and quantification (enabled by the SILAC approach).

Figure 23

Detection of protein persulfidation by differential peptide tagging. An engineered maleimide with a peptide arm and a molecular mass of ∼2 kDa (MalP) reacts with both thiols and persulfides. DTT removes the mixed disulfides formed with persulfides and increases the electrophoretic mobility of the protein.

Figure 24

Cyanoacetic acid-based tag-switch method for persulfide labeling. (A) Both persulfides and thiols are initially blocked with MSBT. The product with a persulfidated cysteine is a mixed aromatic disulfide that can be nucleophilically attacked by a cyanoacetic acid–based probe causing a tag-switch. (B) Three different tags are attached to cyanoacetic acid: BODIPY (CN-BOT), Cy3 (CN-Cy3), and biotin (CN-biotin).

Figure 25

Overview of methodological approaches for total sulfane sulfur detection. Sulfane sulfur compounds release H₂S upon DTT treatment. Sulfane sulfur can be detected by the cold cyanolysis method; samples are incubated in alkaline (pH 8–10) cyanide solutions to release SCN⁻, which is then quantified spectrophotometrically as a complex with Fe³⁺. Sulfane sulfur can be extracted by triarylphosphines in the form of triarylphosphine sulfide, which can be quantified by isotope dilution MS analysis. Finally, different fluorescence probes (e.g., the SSP series) can detect sulfane sulfur in protein persulfides and low molecultar weight hydropolysulfides in cells (described in greater detail in Chart 28).

Figure 26

Factors that might favor protein persulfidation over trans-nitrosation in the reaction of an S-nitrosothiol (RSNO) with H₂S. (A) Resonance structures of RSNO. Nucleophilic attack on the sulfur is favored in Structure D. (B) Interactions with the protein environment could stabilize the resonance structure D. Positively charged Arg or Lys residues could stabilize the NO moiety, while negatively charged Glu or Asp residues could stabilize the sulfur. In addition electric fields (EF) created by the protein environment could selectively stabilize a resonsance structure, e.g., D, promoting persulfidation and HNO release.

Figure 27

Depersulfidation by thioredoxin (Trx). (A) The structure of human thioredoxin (PDB: 5DQY). (B) Thioredoxin reduces protein persulfides and releases H₂S. Oxidized Trx is reduced by thioredoxin reductase (TrxR) at the expense of NADPH. (C) Two possible mechanisms for protein depersulfidation. Top: The nucleophilic thiol attacks the inner sulfur of the protein persulfide forming a mixed protein-Trx disulfide and releasing H₂S. In the next step the resolving cysteine reduces the mixed disulfide forming fully oxidized Trx. Bottom: Trx undergoes persulfidation, forming Trx persulfide, which is reduced by the resolving cysteine with concomitant release of H₂S.

Figure 28

H₂S may regulate cellular antioxidant defenses and prevent senescence by persulfidation of Keap1. In the cytosol, Keap1 represses Nrf-2 signaling by binding to it. Bound Nrf-2 is subjected to polyubiquitination and proteasomal degradation. Persulfidation of cysteine residues in Keap1 induces a conformational change, which results in Nrf-2 release. Nrf-2 translocates to the nucleus where it upregulates the expression of various antioxidant defense genes.

Figure 29

Possible role of H₂S in endoplasmic reticulum (ER) stress. Under ER stress, the activity of the transcription factor ATF4 is increased, resulting in the upregulation of CSE and the cystine transporter Slc7a11. The subsequent increased production of H₂S leads to the persulfidation of protein tyrosine phosphatase 1B (PTP1B) and consequently to an increase in pERK phosphorylation. pERK activation results in global inhibition of protein translation by activation of eukaryotic translation initiation factor 2α (eIF2α). eIF2α induces ATF4 nuclear translocation. The increased production of H₂S during ER stress also results in persulfidation of glycolytic and tricarboxylic acid (TCA) cycle enzymes.

Figure 30

Persulfidation of NF-κB may regulate apoptosis. The proinflammatory cytokine TNFα, involved in the control of inflammatory reactions, stimulates CSE transcription by activating the SP1 transcription factor, resulting in increased H₂S levels. H₂S induces persulfidation of Cys38 in the p65 subunit of NF-κB, enhancing the binding of NF-κB subunits to the coactivator RPS3. The activator complex then migrates to the nucleus where it upregulates the expression of several antiapoptotic genes. TNFα, tumor necrosis factor α; CSE, cystathionine γ lyase; p50 and p65 subunits of NF-κB; RPS3, ribosomal protein S3; SP1, specificity protein-1.

Figure 31

Possible regulatory role of H₂S on the catalytic activity of parkin. (A) In healthy subjects, parkin, a E3 ubiquitin ligase, is persulfidated, which increases its enzymatic activity. This leads to ubiquitination of diverse substrates and their subsequent proteasomal degradation. (B) In patients with Parkinson’s disease, parkin is S-nitrosated. The decreased catalytic activity results in protein aggregation, accumulation of toxic proteins, and cell death.

Figure 32

Some possible physiological roles of H₂S in neurodegenerative disorders. H₂S has been shown to be involved in pathogenesis of Huntington’s, Alzheimer’s, and Parkinson’s diseases, spinocerebellar ataxia, and traumatic brain injury. In healthy subjects, the expression of CSE is regulated by SP1 and ATF4 transcription factors. In Huntington’s disease, abnormal mutated huntingtin (mHtt) protein binds to SP1 and inhibits its activity. Reduced CSE expression results in oxidative stress that subsequently affects ATF4 expression. H₂S also inhibits the production of amyloid beta (Aβ) at different catalytic steps of Aβ. The mature isoform of APP is cleaved by β- and γ-secretases forming Aβ. H₂S interferes with APP maturation and inhibits the activity of β- and γ-secretases leading to the decreased production of Aβ. In Parkinson’s disease, H₂S induces persulfidation of parkin and increases E3 ubiquitin ligase activity. H₂S has beneficial effects in spinocerebellar ataxia type 3, where it regulates protein persulfidation and improves SCA3-associated tissue degeneration. In traumatic brain injury H₂S exerts antiapoptotic effects and down-regulates the expression of autophagy-related proteins, reduces brain edema and improves the recovery of motor and cognitive dysfunction.

Figure 33

Possible signaling roles of H₂S in the vascular system. At a sensory nerve ending, H₂S interacts with NO^• to give HNO. HNO activates TRPA1 channels; this results in Ca²⁺ influx and subsequent release of calcitonin gene-related peptide (CGRP). Binding of CGRP to its receptor on vascular smooth muscle cells activates the adenylate cyclase and the cyclic adenosine monophosphate (cAMP)-controlled downstream signaling pathways. As a result of elevated cAMP, protein kinase A (PKA) is activated and could potentially increase the activity of eNOS. Persulfidation of Cys443 on eNOS increases the activity of the enzyme as well as its ability to be phosphorylated, which results in increased production of NO^• and activation of soluble guanylate cyclase (sGC). H₂S potentiates the binding of NO^• to sGC by reducing ferric heme to the ferrous state. Degradation of cGMP by phosphodiesterase (PDE) is prevented by H₂S. These effects result in vasodilation of smooth muscle cells. Persulfidation of Cys43 on K_ATP channel enhances its activity, resulting in the influx of K⁺ and hyperpolarization of vascular smooth muscle cells. H₂S also plays a role in angiogenesis. Binding of VEGF to its receptor on endothelial cells induces the production of H₂O₂ by activating NADPH oxidase (Nox). H₂O₂ supposedly increases CSE expression leading in turn, to increased H₂S production. H₂S stimulates activation of the Akt signaling cascade, which results in the phosphorylation of eNOS, increasing its activity. NO^• acts as a pro-angiogenic factor.

Figure 34

Possible H₂S effects on glomus cells of carotid bodies under hypoxic conditions. Under hypoxic conditions the levels of H₂S in glomus cells of carotid bodies are elevated. This could be a result of decreased phosphorylation of CSE due to the lack of CO produced by heme oxygenase-2 (HO-2). The lack of CO results in the inhibition of cyclic guanosine monophosphate (cGMP)-stimulated activation of phosphokinase G. The overall increase in H₂S levels activates the L-type Ca²⁺ and T-type voltage-gated Ca²⁺ channels and mobilizes Ca²⁺ from the endoplasmic reticulum (ER).

Chart 1

Measurement of H₂S through Formation of Methylene Blue

Chart 2

Reaction of Monobromobimane with H₂S to form Dibimane Sulfide

Chart 3. Examples of Fluorescent Probes for H₂S Detection Based on the Reduction of Azide or Nitro Groups^a

^a(A) Reduction of azide groups in rhodamine (top), dansyl (middle), and naphthalimide (bottom) scaffolds. (B) Reduction of nitro group in the naphthalimide scaffold.

Chart 4. Examples of Fluorescent Probes for H₂S Detection Containing Two Electrophilic Centers^a

^a(A) Probe containing an aldehyde and an acrylate group on a triaryl pyrazoline scaffold. (B) Probe containing an activated disulfide on a fluorescein scaffold.

Chart 5

Example of a Probe for H₂S Detection Based on the Release of Copper Sulfide from a Cyclen and Fluorescein Derivative

Chart 6. Reactions Catalyzed by CBS^a

^aReaction 1 generates cystathionine in the canonical transsulfuration pathway. Reactions 2–4 generate H₂S from cysteine and/or homocysteine, and reaction 5 produces Cys-SSH from cystine.

Chart 7. Reaction Mechanism of CBS and Structures of Key Intermediates^a

^a(A) A minimal mechanism is shown for the β-replacement of serine by homocysteine to generate cystathionine and water. Structures of the carbanion (B) and aminoacrylate (C) intermediates trapped in Drosophila CBS (PDB: 2PC4 and 2PC3) are shown. The numbering of residues shown in parentheses in B and C are for human CBS. The corresponding residues in the fly protein are Lys88, Ser116, Asn118, and Ser318, respectively. An sp² hybridized α carbon and sp² hybridized α and β carbons are seen in the carbanion and aminoacrylate intermediates, respectively. Lys119 undergoes a major positional shift in the aminoacrylate intermediate where it is no longer required to stabilize the carbanion.

Chart 8. Binding of NO^• and CO to Ferrous CBS^a

^aBinding of NO^• is fast and predicted to occur via displacement of the His65 ligand. Binding of CO is slow and limited by the slow dissociation of the Cys52 ligand.

Chart 9. Reactions Catalyzed by CSE^a

^aThe first reaction is the cleavage of cystathionine to cysteine, α-ketobutyrate (α-KB), and ammonia in the canonical transsulfuration pathway. The next five reactions produce H₂S, while the last two generate the corresponding persulfides from cystine and homocystine.

Chart 10. Outline of CSE Mechanism Illustrated for the γ-Elimination of Homocysteine (Red Box) or the γ-Replacement of Homocysteine by a Second mole of the Same Substrate (Blue Box)^a

^aHcy-S⁻ denotes homocysteine. The first few steps until intermediate I are common to both pathways.

Chart 11. Reaction Catalyzed by MST^a

^a(A) 3-Mercatopyruvate is synthesized by L-cysteine aminotransferase (CAT), which requires α-ketoglutarate as a cosubstrate. In the first half reaction, MST transfers the sulfur atom from 3-mercaptopyruvate to an active site cysteine forming a Cys-SSH intermediate. (B) In the second half reaction, the outer sulfur from Cys-SSH is transferred to a small molecule thiol acceptor (RSH) or to thioredoxin (Trx) and subsequently released as H₂S. (C) 3-Mercatopyruvate can be synthesized from D-cysteine via the action of D-amino acid oxidase (DAO).

Chart 12. Overview of the Reaction Catalyzed by SQR^a

^aIn the sulfurtransferase steps, HS⁻ attacks the disulfide bond in SQR forming a persulfide at Cys379 and the sulfane sulfur (in red) is transferred to an acceptor (GSH, sulfite, sulfide, or cyanide). In the electron transfer steps, two electrons are transferred from HS⁻ through the disulfide to FAD and then to CoQ.

Chart 13. Postulated Reaction Mechanism of SQR^a

^aNucleophilic attack of HS⁻ on the active site disulfide results in the formation of a Cys-SSH intermediate at Cys379 and a charge transfer (CT) complex, which collapses to a postulated 4a adduct. A sulfur acceptor viz. GSH or sulfite moves the sulfane sulfur from the active site, forming GSSH or thiosulfate (S₂O₃²⁻), respectively, and restoring the active site disulfide. Electron transfer from the reduced flavin, FADH₂, to CoQ completes the catalytic cycle.

Chart 14. Postulated Reaction Mechanism of PDO^a

^aBinding of GSSH to the resting enzyme [1] creates a binding site for O₂ [2]. Formation of a superoxo-Fe^III [3] in resonance with a biradical Fe^II species [4] leads to formation of a cyclic peroxo-Fe^II species [5]. Cleavage of the O–O bond gives [6]. Alternatively, cleavage of the Fe–O bond gives [7]. Binding of H₂O [8], sets up hydrolysis and formation of the product, sulfite. Alternatively, the water that remained coordinated to the metal center could be used for the final hydrolysis step.

Chart 15. Reactions Catalyzed by Rhodanese^a

^aRhodanese exhibits varied sulfur transferase activities including: (A) thiosulfate:cyanide sulfurtransferase, (B) GSSH:sulfite sulfurtransferase, and (C) thiosulfate:GSH sulfurtransferase. E-SSH denotes the enzyme-bound Cys-SSH intermediate. The red color traces the fate of the sulfur from the donor to the acceptor.

Chart 16. Molybdopterin Cofactor in Sulfite Oxidase and Redox Changes during the Catalytic Cycle^a

^a(A) Attack of sulfite on an oxo/hydroxyl ligand reduces the molybdenum ion. (B) The reaction cycle of sulfite oxidase involves an initial two-electron reduction of the molybdenum center, which is subsequently oxidized in two one-electron steps via intramolecular electron transfer to the heme. The latter in turn, transfers electrons to the heme in cytochrome c (cyt c) in an intermolecular process.

Chart 17. Interaction of H₂S with Cytochrome c Oxidase^a

^a(A) At low concentrations, H₂S binds to ferric heme a₃ and reduces it with concomitant formation of HS^• which can react with either another molecule of H₂S or with oxygen. Reduction of heme a₃ leads to an increase in oxygen consumption. (B) At moderate levels, H₂S interacts with the Cu_B⁺ center forming a stable Cu_B–SH⁻ complex, which is difficult to oxidize. Cu_B⁺ is formed by electron transfer from ferrous heme a₃ or by a direct reduction by H₂S. At higher levels of H₂S, the Cu_B–SH⁻ complex induces a conformational change and causes further binding of H₂S to ferric heme a₃. (C) Alternatively, H₂S can reduce cytochrome c thereby increasing CcO reduction and respiration.

Chart 18. Sulfhemoglobin Formation^a

^a(A) One of the proposed structures of sulfheme. (B) The mechanism of sulfheme formation is not fully understood (as denoted by the question marks) but starts with compound I or II reacting with H₂S and results in sulfur being incorporated into the porphyrin ring.

Chart 19. Minimal Reaction Mechanism for Ferric Globin-Dependent Sulfide Oxidation to Thiosulfate and Iron-Bound Hydropolysulfides^a

^aDetails of the oxidation chemistry that lead to the products have been omitted for clarity.

Chart 20. Interaction of H₂S with NO^• and Its Metabolites^a

^aNO^• signals via the classical soluble guanylate cyclase (sGC)/cyclic GMP (cGMP) cascade. H₂S can reduce sGC to increase NO^• binding and cGMP production. cGMP is deactivated by phosphodiesterase 5 (PDE), an enzyme that is inhibited by H₂S. Oxidation of NO^• to nitrosonium (NO⁺) ion leads to modification of cysteine residues and formation of S-nitrosothiols (RSNO), a process that H₂S can facilitate. NO^• can be reduced by H₂S to form HNO. HNO activates the release of calcitonin gene-related peptide (CGRP), a vasodilator, but HNO can also be trapped by H₂S. NO^• is oxidized to nitrite, which can be reduced back to NO^•, a process that H₂S can facilitate. NO^• reacts with superoxide to form peroxynitrite (ONOO⁻). H₂S can scavenge ONOO⁻.

Chart 21

Reaction Paths for HSNO Generation (1–3) and Its Biologically Relevant Reactions (4–6)

Chart 22. Proposed Reaction Mechanism for H₂S-Assisted Nitrite Reduction Catalyzed by an Iron Porphyrin Compound^a

^aPathway A, which predominates when nitrite is in excess over H₂S, represents a classical oxygen atom transfer; nitrite coordination to ferric heme leads to HSOH and [Fe²⁺(NO)] ↔ [Fe³⁺(NO⁻)], which releases HNO slowly. Pathway B predominates when H₂S is in excess over nitrite; reduction of ferric heme by H₂S is followed by nitrite reduction to [Fe³⁺(NO)] ↔ [Fe²⁺(NO⁺)] species, which is scavenged by HS⁻ giving HSNO. Either free or coordinated HSNO causes transnitrosation of protein thiols or, in the reaction with H₂S, generates HNO.

Chart 23

Synthetic Strategies for the Preparation of Low Molecular Weight Persulfides (A) and Structures of Some of the Persulfides Prepared following These Synthetic Routes (B)

Chart 24

Synthesis of an Acyl-Protected Disulfide of Penicillamine (A) and Its Rearrangement to N-Methoxycarbonyl Penicillamine Persulfide via S- to N-Methoxycarbonyl Transfer at pH 7.4 (B)

Chart 25. Strategies for the in Situ Preparation of LMW Persulfides^a

^a(A) Cysteine and glutathione persulfides can be prepared by the reaction of H₂S with cystine and glutathione disulfide, respectively. (B) Cysteine persulfide can be generated from cystine and CSE or CBS. (C) Rhodanese can be used to transfer sulfur to glutathione. (D) Glutathione reductase (GR) uses electrons from NADPH to reduce glutathione trisulfide to persulfide and GSH.

Chart 26. Reactions of Persulfides with Electrophiles^a

^a(A) Reactions with thiol alkylating agents. (B) Reactions with disulfides. (C) Reactions with methylmercury and nitroguanosine 3′,5′-cyclic monophosphate.

Chart 27. Protein Persulfidation Can Protect Proteins from Overoxidation^a

^aA thiol can be oxidized to a sulfenic acid. The latter can be reduced back to thiol or be further oxidized to sulfonate, an irreversible modification. Persulfides, if exposed further to oxidants, will form S-sulfocysteines (−SSO₃⁻). Enzymes such as thioredoxin can reduce the S–S bonds and restore the native thiol.

Chart 28. Fluorescent Probes for Sulfane Sulfur^a

^a(A) Internal cyclization and fluorophore release following the initial reaction of the probe with sulfane sulfur compounds. (B) Structures of the synthetic probes that exhibit this type of chemical reactivity.

Chart 29. Electrophilic Probes that Release a Fluorophore upon Reaction with Inorganic Polysulfides^a

^a(A) Reactions leading to fluorophore release. (B) Structures of the synthetic probes that exhibit this type of reactivity. (C) FRET-based probe for the simultaneous detection of H₂S and sulfane sulfur-containing species.

Chart 30

Ratiometric Near-IR Fluorescence Probe for Cysteine Persulfide Detection (A) and FRET Probe Designed for Persulfide Detection (B)

Chart 31. Isotope Dilution Mass Spectrometry Approach for the Detection of Sulfane Sulfur Using Substituted Phosphines^a

^aThe reaction of triarylphosphine (P2) with sulfane sulfur compounds. After incubation of P2 with cell or tissue samples to form PS2, PS1 (¹³C-labeled triarylphosphine sulfide) is added as internal standard and the samples analyzed by MS.

Chart 32. Reactions of Inorganic Polysulfides with Proteins^a

^a(A) Inorganic polysulfides can react as electrophiles with a protein thiolate. The resulting polythiolated cysteine can have different numbers of S atoms and can in turn, be attacked by a proximal cysteine forming a family of products, e.g., intramolecular disulfides, trisulfides, etc. (B) Inorganic polysulfides can react as nucleophiles with an intramolecular protein disulfide forming thiol and polythiolated cysteine.

Chart 33. Active Principles of Garlic and the Mechanisms for LMW Persulfide Generation from Them^a

^a(A) Allicin (diallyl thiosulfinate) is rapidly metabolized in aqueous solutions into diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and ajoene. (B) Glutathione-promoted decomposition of DADS and generation of allylpersulfide, glutathione persulfide (GSSH), and H₂S.

Chart 34

Base-Promoted Persulfide Generation from Cystine

Chart 35

Tautomerization of a Persulfide to a Thiosulfoxide as a Postulated Mechanism for Transpersulfidation

Chart 36. Glutaredoxin-Catalyzed Protein Depersulfidation^a

^aGlutaredoxin (Grx) reduces a protein persulfide, oxidized Grx is reduced by glutathione (GSH), and GSSG is reduced by glutathione reductase (GR) at the expense of NADPH.