Hydrogen Sulfide and Hemeproteins: Knowledge and Mysteries

Abstract

Historically, hydrogen sulfide (H₂S) has been regarded as a poisonous gas, with a wide spectrum of toxic effects. However, like ·NO and CO, H₂S is now referred to as a signaling gas involved in numerous physiological processes. The list of reports highlighting the physiological effects of H₂S is rapidly expanding and several drug candidates are now being developed. As with ·NO and CO, not a single H₂S target responsible for all the biological effects has been found till now. Nevertheless, it has been suggested that H₂S can bind to hemeproteins, inducing different responses that can mediate its effects. For instance, the interaction of H₂S with cytochrome c oxidase has been associated with the activation of the ATP-sensitive potassium channels, regulating muscle relaxation. Inhibition of cytochrome c oxidase by H₂S has also been related to inducing a hibernation-like state. Although H₂S might induce these effects by interacting with hemeproteins, the mechanisms underlying these interactions are obscure. Therefore, in this review we discuss the current state of knowledge about the interaction of H₂S with vertebrate and invertebrate hemeproteins and postulate a generalized mechanism. Our goal is to stimulate further research aimed at evaluating plausible mechanisms that explain H₂S reactivity with hemeproteins. Antioxid. Redox Signal. 15, 393–404.

Introduction

Hydrogen sulfide (H₂S) is a well-known poisonous gas whose cytotoxic effects have been studied for >300 years (65). Under physiological conditions, ∼30% of H₂S is undissociated and ∼70% is dissociated to hydrosulfide ion (pKa of 6.8). H₂S is also soluble in water and plasma (1 g in 242 ml at 20°C) and it can penetrate cells of all types by simple diffusion (34). It is this property that makes H₂S a broad-spectrum toxicant. Interestingly, the discovery that the human body naturally produces H₂S has dramatically changed the reputation of this gas from a toxic pollutant to a biologically relevant molecule. H₂S is now considered to be an important physiological mediator with a wide variety of roles, including regulation of neuronal activity and muscle relaxation, which are associated with the activation of N-methyl-D-aspartate receptors and ATP-sensitive potassium (K_ATP) channels, respectively (5, 33, 34, 38, 45, 77). Activation of the K_ATP channels has been related to the interaction of H₂S with several cysteine residues within the complex (27). It has also been suggested that K_ATP activation might be mediated by the interaction of H₂S with hemeproteins such as cytochrome c oxidase (CcO), myoglobin (Mb), and hemoglobin (Hb), which decreases cellular ATP and activates K_ATP channels (19, 30, 75, 77).

CcO is one of the key enzymes responsible for cellular respiration, whereas Mb and Hb are involved in oxygen (O₂) transport. The reaction between CcO and H₂S induces modification of the heme a₃ and Cu_B centers in the enzyme, reversibly inhibiting its activity and reducing ATP production (18, 19, 29, 53–55). The reduction in cellular ATP can then activate the K_ATP channels, which are otherwise blocked by ATP. In Mb and Hb, H₂S can also bind and modify the heme. The resulting derivatives, named sulfmyoglobin, sulfhemoglobin, or sulfheme species (6–9, 16), have lower O₂ affinity, thereby reducing O₂ transport and ATP production in the mitochondria, which can in principle, also activate the K_ATP channels.

The interaction of H₂S with CcO has also been implicated in the stimulation of a hibernation-like state in animals that do not normally hibernate (18, 44, 48). In this regard, it has been shown that H₂S reduces O₂ consumption and metabolic activity in mice, allowing the rodent to survive low O₂ concentrations that are otherwise lethal to them. In particular, the reduction in metabolic activity has been related to the decrease of cellular ATP induced by the interaction of H₂S with CcO and Hb/Mb. Based on these findings researchers are now investigating the possibility of injecting H₂S into patients with conditions related to insufficient blood supply to temporarily reduce metabolic activity and O₂ demand until they receive the appropriate treatment (48). Moreover, a number of drug candidates that mimic H₂S effects are now in development (46, 47, 50, 75). Therefore, understanding the mechanisms underlying the interaction of H₂S with its targets is crucial for the successful development of H₂S-based drugs. In fact, in the presence of sulfide, hemeproteins show different chemical reactivities. H₂S coordinates preferentially to the open sixth position of ferric iron (Fe^III) (34, 40, 61). Upon coordination, the gas can form a stable low spin Fe^III-SH₂ complex or it can reduce the ferric iron producing an unstable, Fe^II-SH₂ intermediate, which is rapidly converted to the unligated ferrous iron (Fe^II) (40, 61). H₂S can also react with oxy-hexacoordinated hemes (Fe^II-O₂) to form Fe^III-SH₂, most probably by nucleophilic displacement of the bound superoxide (40). Reaction of H₂S with Fe^II-O₂ can also modify the heme active center producing sulfheme complexes.

In CcO, H₂S modifies enzyme activity by binding and reducing the metal centers, whereas in Mb and Hb, H₂S reacts with the oxy form of the proteins (Fe^II-O₂) generating the sulfheme derivative. Why does H₂S react differently with these hemeproteins? What is the role of the heme iron oxidation state? Further, how does H₂S interact with hemeproteins to generate different intermediates and products? Remarkably, interactions of H₂S with hemeproteins have been recognized and studied for many years in invertebrate organisms (2, 26, 40, 41, 78, 80), making these systems excellent models to understand the reactivity of H₂S with other hemeproteins. In this review, we will evaluate the interaction of H₂S with vertebrate hemeproteins and correlate these interactions using the invertebrate hemeproteins as models. The metabolism of H₂S is briefly described, followed by an overview of sulfide reactivity with hemeproteins. Importantly, the attacking and coordinated sulfide species (i.e., whether it is H₂S or the hydrosulfide ion) is unknown. Since at physiological pH, the predominant form is the hydrosulfide ion (HS⁻), it has been suggested that it might be the attacking and the bound species. However, in Mb and other hemeproteins, sulfide binding decreases at alkaline pH, suggesting that the undissociated H₂S might be the attacking species in some hemeproteins (12, 40). In fact, the slow association rate observed at neutral pH has been ascribed to the rate-limiting protonation of HS⁻ to H₂S (40). Like H₂O, once H₂S binds to the heme, an equilibrium between Fe^III-SH⁻ and Fe^III-SH₂ can occur. Although it has not been possible to determine which form of sulfide is coordinated in hemeproteins, the term “H₂S” is generally used. Hence, in the remainder of this review, H₂S refers to the combination of the undissociated species and the hydrosulfide anion.

Overview of H₂S Metabolism in Humans

Endogenous H₂S in humans is produced directly by two pyridoxal phosphate (PLP)-dependent enzymes, cystathionine β-synthase (CBS, EC 4.2.1.22) and cystathionine γ-lyase (CSE, EC 4.4.1.1), and indirectly by 3-mercaptopyruvate sulfur transferase (3MST, EC 2.8.1.2) (33, 34, 38, 71). The catalytic core of CBS is constituted by two main domains, a PLP-containing domain and a heme-containing domain (Fig. 1A). CBS produces H₂S at the PLP active site through multiple reactions but preferably via condensation of cysteine and homocysteine (Fig. 1B) (33). The heme in CBS is a six-coordinate, low spin protoporphyrin IX with cysteine and histidine residues serving as endogenous axial ligands. This heme is believed to have a regulatory function and inhibits CBS activity upon binding CO in the ferrous state or by undergoing a redox-dependent ligand switch (3, 4, 13, 35, 62, 69, 70, 79). Binding of CO in ferrous CBS perturbs the heme environment and this is communicated to the PLP active site, resulting in a shift in the tautomeric equilibrium of PLP from the active ketoenamine to the inactive enolimine state (35, 79). CSE is mainly present in the peripheral tissues and under normal conditions, it catalyzes the α,β-elimination of cysteine, producing H₂S, pyruvate, and NH₃ (Fig. 1B) (17, 33, 34, 38). On the other hand, 3MST catalyzes the desulfuration of 3-mercaptopyruvate, which is produced from cysteine by cysteine aminotransferase. Unlike CBS and CSE, the final product of the 3MST enzymatic reaction is persulfide, not H₂S (33). H₂S is expected to be generated from the persulfide product only under reducing conditions (33, 34, 38).

FIG. 1.

Structure of CBS and H₂S generation. (A) Structure of the pyridoxal phosphate and heme domains of CBS generated from PDB file 1M54. (B) Enzymatic pathways of H₂S production in humans. The gas is produced by CBS, CSE, and MST from cysteine. H₂S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; MST, mercaptopyruvate sulfur transferase. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

Once generated by either enzymatic pathway, H₂S acts on target cells to either regulate neural or muscle activities (Fig. 2) (31, 33, 34, 38). In the brain, H₂S has been suggested to act as a neuromodulator and a neuroprotector by facilitating the induction of hippocampal long-term potentiation and protecting the neurons from oxidative stress. These effects have been related to the activation of N-methyl-D-aspartate receptors and to an increase in glutathione levels (34, 38). As a muscle relaxant, H₂S dilates blood vessels as well as gastrointestinal, pulmonary, and nasal tissues and is also involved in smooth muscle relaxation of the human penile corpus cavernosum tissue (33, 34, 38, 45, 77). The muscle relaxation effect has been attributed to opening of the K_ATP channels. It has also been shown that at low O₂ levels, inhibition of CcO by H₂S stimulates muscle relaxation directly without activating the K_ATP channels (39).

FIG. 2.

Schematic representation of H₂S biosynthesis, biological effects, and degradation. One of the H₂S generating enzymes, CBS, is a hemeprotein. H₂S can act on the target cell or on the same cell by activating the K_ATP channels, a process that might involve sulfide interaction with hemeproteins. H₂S can also activate of N-methyl-D-aspartate receptors, regulating in turn neural activity. H₂S can be degraded to other, less toxic compounds through its reactivity with hemoglobin (Hb) or myoglobin (Mb). For instance, degradation of sulfhemoglobin and sulfmyoglobin by the physiologic turnover of red blood cells is considered to be one of the pathways for H₂S removal. The figure only highlights the heme-dependent pathways that might play a role in H₂S degradation. K_ATP, ATP-sensitive potassium. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

H₂S is mainly degraded in the mitochondria through a series of oxidation reactions (33, 34, 38), which convert the gas to sulfite (SO₃⁻²), thiosulfate (S₂O₃⁻²), and sulfate (SO₄⁻²). In addition, H₂S can be catabolized by cytosolic methylation to dimethylsulfide (34). Interaction of H₂S with Mb and Hb to form the sulfheme complex has also been suggested to be one of pathways for H₂S removal (34, 46, 75).

Interaction of H₂S with Vertebrate Hemeproteins

Cytochrome c oxidase

CcO is involved in the final step of aerobic respiration and reduces O₂ to water. It contains two hemes, heme a and heme a₃, and two copper centers, Cu_A and Cu_B (Fig. 3). The catalytic center comprises the heme a₃, the Cu_B, and a tyrosine residue, which is coordinated to Cu_B. Physiologically, the complex receives electrons from cytochrome c and transfers them through Cu_A, heme a, and the heme a₃-Cu_B center. The reduced ferrous (Fe^II) heme a₃ binds O₂ and transfers electrons to the ligand, reducing O₂ to water. During this process, protons are translocated across the mitochondrial membrane, establishing an electrochemical potential that is used by another enzyme to synthesize ATP.

FIG. 3.

Structure of the CcO catalytic center and its reactivity with H₂S. (A) The heme a₃ and Cu_B catalytic centers of CcO under normal conditions and upon inhibition by H₂S. Schemes for H₂S interaction with CcO as suggested by Nicholls (29, 52, 53, 55) (B) and by Collman (C) (17). At low to moderate concentrations, H₂S interacts with the heme a₃/Cu_B binuclear center, modifying CcO activity and at higher concentrations, H₂S binds directly to the ferric heme a₃ group inhibiting CcO. CcO, Cytochrome c oxidase. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

The effects of sulfide on the activity of CcO were known since the 18th century; however, the inhibition of enzyme by H₂S was first described by Keilin in 1929, who observed a substantial reduction of CcO activity after exposure of different tissues to the gas (36). Using various spectroscopic approaches, Nicholls and others later demonstrated that H₂S binds directly to the ferric (Fe^III) heme a₃ center and completely inhibits CcO activity (Fig. 3A) (28, 29, 53–55). However, they observed that at low (nearly stoichiometric) H₂S concentrations, the gas stimulates O₂ consumption without having an inhibitory effect and that >1 mol of H₂S was required for complete inhibition of the enzyme. Based on these findings, a generalized mechanism was proposed, which explained the interaction of H₂S with the resting enzyme as depicted in Figure 3B. At low sulfide levels (1:1 stoichiometry) H₂S binds and reduces the ferric heme a₃ center in a reaction that results in the formation of a ferrous heme a₃-SH₂ intermediate, release of sulfide (as an SH radical or as elemental sulfur), and O₂ uptake. They suggested that under these circumstances, H₂S could act as a substrate instead of an inhibitor (54, 55). At moderate concentrations (1:2 stoichiometry), H₂S also coordinates and reduces the Cu_B center with the concomitant formation of a stable Cu_B-SH₂ moiety. It was presumed that the heme a₃-Cu_B-SH₂ center adopts a new conformational state, which is unable to form a stable complex with O₂. It was therefore suggested that at higher H₂S concentrations (1:3 stoichiometry), full inhibition of CcO results because H₂S binds tightly to ferric heme a₃, either because heme a₃ is in a different conformational state or because the Cu_B-SH₂ complex restrains the electron transfer process to the ferric heme. Recently, Collman and coworkers confirmed that at low concentrations, the gas indeed reduces heme a₃ without inducing inhibition (18). They also observed that at moderate H₂S concentrations, the gas binds to the ferrous heme a₃ of CcO with the immediate formation of a ferrous heme a₃-SH₂ derivative that reversibly inhibits CcO activity (Fig. 3C) (18). In the presence of O₂, H₂S is rapidly displaced restoring CcO activity.

These models beg the question as to how at low H₂S concentrations, the gas reduces the metal centers of CcO without inhibiting its activity, whereas at higher concentrations complete inhibition is observed. We have demonstrated recently that heme reduction is greatly enhanced at high H₂S concentrations and in proteins having proton acceptor groups in the vicinity of the ligand (61). Hence, to propose a plausible mechanism to explain the binding and inhibition kinetics of H₂S with CcO, H₂S concentration and the polarity of the ligand binding site should be considered. We propose that at low sulfide concentration, the polar environment near heme a₃, which includes the Cu_B center and a tyrosine residue, may stimulate heme a₃ reduction by H₂S. At higher concentrations (1:3 stoichiometry), H₂S also binds and reduces the Cu_B and heme a₃ centers, forming a stable Cu_B-SH₂ moiety and the unstable heme Fe^II-SH₂ inhibitory complex, as suggested by Collman et al. (18). At this stage, reduction of Cu_B and heme a₃ may be facilitated by the presence of slight H₂S excess. However, since an excess of H₂S enhances heme reduction, exhaustion of the gas might limit heme a₃ reduction, stabilizing in turn the final heme a₃ Fe^III-SH₂ inhibitory species. Based on this model one can hypothesize that at moderate concentrations, H₂S can exert protective effects in mammals. Under these conditions the affinity of heme a₃ for O₂ decreases, diminishing in turn cellular ATP, which can in principle stimulate muscle relaxation.

Hb and Mb

Vertebrate Hb and Mb are globular hemeproteins whose physiological functions are related to their ability to bind molecular O₂. In mammals, Hb consists of four globular chains, each containing a heme group. In contrast, Mb is a monomeric globular protein with a single heme group. In both hemeproteins, O₂ binds directly to the ferrous iron. O₂ binding is stabilized in part by a neighboring histidine residue (Fig. 4) and is later released for utilization in mitochondrial ATP production. Both Hb and Mb bind H₂S in the ferric state as a heme ligand. However, the affinity is very low and heme reduction by H₂S is rapidly observed with the concomitant formation of the deoxy Fe^II and/or oxy Fe^II-O₂ derivatives.

FIG. 4.

The active site of Mb with bound oxygen (left) and after exposure to H₂S (right). The final sulfheme product is a modified chlorin-type heme with a sulfur atom incorporated into one of the pyrrole rings. The structures were generated using PDB files 1MBO (left) and 1YMC (right), respectively. In Hbs, the amino acids are arranged in six to eight helical segments that are labeled A to H with the first residue of segment A being A1. In the figure, HisE7 refers to the histidine residue at position 7 of helix E, which in human Hb and Mb stabilizes O₂ through hydrogen bonding interactions.

The reaction of H₂S with Hb and Mb was first recognized in 1863 by Hoppe-Seyler, who observed the formation of a green compound when the proteins were exposed to the gas in the presence of O₂ (37, 52). He described these new compounds as sulfhemoglobin and sulfmyoglobin with a characteristic absorption band at ∼618 nm. In 1933, Keillin showed that O₂ was essential for the formation of these compounds and that they were only obtained from the oxy (Fe^II-O₂) and met (Fe^III-OH₂) forms of the proteins (37). However, in the met complex, prior reduction of the heme by H₂S was required to produce the sulfheme derivatives. It was later shown that these sulfhemoglobin and sulfmyoglobin complexes consisted of a chlorin type heme in which one of the pyrrole rings was modified by the incorporation of the sulfur atom across the β-β double bond of the pyrrole “B” (Fig. 4) (6–9, 16). Incorporation of the sulfur atom was suggested to remove electron density from the ferrous iron toward the periphery of the chlorin ring. The delocalization of π electron density away from the iron was supported in part by vibrational analysis of the sulfheme-CO complexes, in which an increase in the stretching frequency of bound CO was observed (6, 14). A decrease in electron density of the “d” iron orbitals decreases the backbonding density form the iron to the antibonding π*orbital of carbon monoxide, increasing in turn the energy of the CO vibrational normal mode. Since O₂ forms a stable complex only with ferrous heme, the electron delocalization away from the iron in the sulfheme chlorin ring was therefore expected to decrease O₂ affinity (14). Subsequent studies demonstrated that, in fact, the sulfheme compounds were able to bind O₂ reversibly but with a much lower affinity than the unmodified proteins and that O₂ binding was dictated by the specific aspect of the overall hemeprotein structure (6, 8, 14). O₂ binding in sulfmyoglobin was shown to be ∼2500-fold lower, whereas binding in sulfhemoglobin was reduced by a factor of ∼135 (14). In Mb and Hb, the final sulfheme product cannot be reconverted to the normal hemeproteins by natural mechanisms in the red cells (8).

Like methemoglobin, which results from the autoxidation of the ferrous iron to the nonfunctional ferric state of the protein, high levels of sulfhemoglobin can be potentially toxic. The inability of both sulfmyoglobin and sulfhemoglobin to bind O₂ effectively can lead to sulfhemoglobinemia, a rare cause of cyanosis induced by drugs such as phenacetin, dapsone, sulphonamides, and metoclopramide, and by exposure to other sulfur-containing compounds (57, 68, 76). However, moderate concentrations of sulfhemoglobin are well tolerated despite the low O₂ binding (1, 25). This has been explained by the observed right shift in the Hb-O₂ dissociation curve, which indicates that although it is more difficult for sulfhemoglobin to bind O₂, it facilitates O₂ delivery to tissues. Also, in most patients, sulfhemoglobinemia has few adverse clinical consequences and are usually resolved spontaneously by normal erythrocyte destruction (1). Therefore, formation of these sulfheme complexes at moderate levels and their destruction by the physiologic turnover of red blood cells can be considered to be one of the pathways for H₂S degradation. It is also reasonable to suggest that the reduced O₂ binding in the sulfheme derivatives decreases ATP synthesis, which in turn could activate the K_ATP channels. Further, the presence of the sulfheme complexes could represent a control mechanism for O₂ uptake by CcO during periods of hibernation.

Importantly, the mechanism by which the sulfheme derivative is formed is unknown. It has been suggested that oxo-ferryl intermediates may be involved in sulfheme formation since sulfhemoglobin and sulfmyoglobin can also be generated with hydrogen peroxide (H₂O₂). It is well known that the reaction of O₂ or H₂O₂ with hemeproteins produces ferryl Compound I, (Fe^IV = O Por^∙+) and Compound II (Fe^IV = O Por) (20–23, 64). Curiously, the reaction of CcO with either O₂ or H₂O₂ also produces oxo-ferryl intermediates, but sulfheme formation has not been detected on this protein. Why do Hb and Mb form sulfheme while CcO does not? As discussed in the next section, we have observed that the histidine residue near the heme in Hb and Mb is essential for sulfheme formation. In CcO, the histidine residues near the heme a₃ center are not adequately orientated to form the sulfheme complex.

Interaction of H₂S with Invertebrate Hemeproteins

From bacteria to plants and animals, hemeproteins are found in virtually all organisms (78). In many invertebrate systems, the reactivity of hemeproteins, and in particular Hbs, toward H₂S has been associated with relevant physiological processes. Marine invertebrates living in sulfide-rich environments represent such an example (2, 40, 41). One of the best studied of these organisms is the clam, Lucina pectinata, and we therefore begin with a description of H₂S interaction with clam Hb.

Hemoglobin I from L. pectinata

The clam L. pectinata lives in sulfide-rich mangroves and it is specifically abundant in the southwest coasts of Puerto Rico. The nutritional needs of this clam, which lacks a mouth and gut, are met by a symbiotic relationship with sulfide-oxidizing bacteria. Bacteria living inside the gill oxidize H₂S in the presence of O₂ and this energy is utilized to synthesize organic nutrients for the invertebrate. Wittenberg and coworkers demonstrated that the protein responsible for delivering H₂S to the bacteria is a hemeprotein called hemoglobin I or HbI (40, 41). HbI is a monomeric protein (Fig. 5A) and it is one of the few known H₂S carriers. Binding of H₂S by HbI not only maintains the symbiotic relationship with the bacteria, but also protects the clam from H₂S toxicity. The affinity of ferric HbI for H₂S is very high and results from fast association (k_on = 2.3 × 10⁵ M⁻¹s⁻¹) and very slow dissociation (k_off = 0.22 × 10⁻³ s⁻¹) rate constants. Structural studies of the HbI active site have shown that this protein has a glutamine residue at the distal ligand binding site, instead of the equivalent histidine typically found in many invertebrate and vertebrate organisms (66). In addition to glutamine, HbI has phenylalanine residues near the heme, generating what is known as the “Phe-cage.”

FIG. 5.

Structure of Lucina pectinata HbI (PDB:1MOH) and its reactivity with H₂S. (A–C) HbI tertiary structure, heme active site, and its reaction with H₂S, respectively. The nomenclature in (B) refers to the positions of the amino acids near the heme. In L. pectinata HbI, the first reaction in (C) dominates at low H₂S concentrations and the second one at higher ligand concentrations.

A close-up of the unusual amino acid composition of the HbI distal ligand binding site is shown in Figure 5B and is believed to be responsible for the high H₂S affinity. Indeed, early spectroscopic studies of HbI with other well-known ligands (such as CO, cyanide, H₂O, and O₂) suggested that glutamine is flexible and controls ligand access to the HbI heme pocket and that hydrogen bonding and other interactions with the Phe-cage contribute to ligand stability (15, 51, 60). Using site-directed mutagenesis, and spectroscopic and theoretical approaches, we have shown that protein fluctuations are required to allow H₂S access to the HbI distal heme site.

Once in the distal heme site, the flexibility of glutamine allows the ligand to bind rapidly to the ferric iron (24, 43, 59–61, 63). The gaseous ligand is stabilized in part by a hydrogen bonding interaction with glutamine (61, 66). H₂S release is dictated by two competing processes involving slow dissociation of H₂S from the ferric adduct and heme iron reduction followed by H₂S liberation (Fig. 5C). The former process dominates at low H₂S concentrations, and the latter at high concentrations (61). Heme reduction is facilitated by the hydrogen bond between glutamine and bound H₂S and replacement of this residue by valine, which disrupts the hydrogen bond, precludes reduction. In addition, the reduction process is greatly enhanced in HbI mutants having proton acceptor groups near the bound H₂S. For instance, replacement of glutamine by histidine results in rapid heme reduction. Further, the HbI glutamine → histidine mutant was the only one in which sulfheme formation was observed after heme reduction. It is plausible that histidine plays an essential role in sulfheme formation and in other hemeproteins lacking a corresponding histidine residue, sulfheme formation is not seen (Table 1). Collectively, these data suggest that the distal-site environment in hemeproteins controls not only H₂S binding and stabilization, but heme reduction and sulfheme formation as well.

Table 1.

Representation of the Different Hemeproteins Evaluated for Sulfheme Formation and Their Corresponding E7 Residues

Protein	E7 Residue	Sulhemea
Horse heart Mb	His	Yes (618 nm)
Human Hb	His	Yes (620 nm)
Lucina Pectinata HbII and HbIII	Gln	No
L. Pectinata Hbl	Gln	No
HbI PheE11Val	Gln	No
HbI PheB10Leu	Gln	No
HbI GlnE7His	His	Yes (622 nm)
Macrobdella decora Hb	His	Yes (620 nm)
Lumbricus terrestris Hb	His	Yes (620 nm)

^aData from ref. (67).

Several factors control H₂S reactivity with hemeproteins: (i) accessibility of H₂S to the heme cavity, (ii) H₂S concentration, which influences heme reduction, (iii) polarity of the distal environment surrounding the bound H₂S, and (iv) orientation of the distal side residues. We have proposed a general scheme to describe the interaction of H₂S with hemeproteins (Fig. 6). At low H₂S concentrations (3 to 10 molar excess), hemeproteins with a relatively open distal pocket will react with H₂S readily forming the heme-SH₂ complex (Fe^III-SH₂). Sulfide release is then dictated by two competing processes involving simple H₂S dissociation and heme iron reduction. Hemeproteins with low polarity environments in the vicinity of the iron, form a stable heme-SH₂ species and sulfide release is dictated by slow H₂S dissociation without inducing significant heme reduction. Hemeproteins in which active-site residues are not oriented to form strong hydrogen bonding interactions with H₂S also show the same reactivity. In contrast, hemeproteins with hydrogen bond acceptor groups, oriented to favor strong interactions with the bound ligand, can stimulate heme reduction. As in HbI, strong hydrogen bond interactions between the hemeproteins and the bound H₂S can facilitate rapid deprotonation of H₂S, stimulating formation of an Fe^II-SH· intermediate by one electron transfer from the Fe^III-SH⁻ species. Reduction of the heme by the Fe^III-SH⁻ species is supported by the fact that in model porphyrinate systems, coordination of HS⁻ to ferric iron produces Fe^II high spin derivatives (58). In the presence of a slight excess of H₂S, the latter can react with the Fe^II-SH· radical intermediate producing deoxy heme Fe^II and a polysulfide species (61). This is also supported by the observation that the interaction of ·SH radicals with H₂S, in the presence of slight sulfide excess, produces polysulfide and H₂S compounds, and that at higher concentrations, these polysulfides can produce elemental sulfur (73). We also suggest that following heme reduction and in the presence of O₂, proteins having a histidine residue in their heme environment can promote sulfheme formation. In fact, we have recently evaluated the role of histidine in sulfheme formation by introducing point mutations in the L. pectinata HbI heme pocket to mimic the distal site of Mb and to identify the residues involved in sulfheme formation (67). Formation of the sulfheme product was monitored by the characteristic 620 nm band and by resonance Raman spectroscopy. Sulfheme production was only observed in the HbI glutamine → histidine mutant and in proteins having histidine in their heme active site, including human Hb and Mb and the giant Hbs from Macrobdella decora and Lumbricus terrestris (67). The 620 nm band is only observed after addition of H₂S to the proteins and is assigned to the sulfheme derivative. We hypothesize that after heme reduction, the ferrous iron coordinates O₂ and the histidine residue stabilizes the peroxo or the oxo-ferryl intermediates, which in the presence of H₂S stimulates sulfheme formation (Fig. 7).

FIG. 6.

Generalized reactions for H₂S reactivity with hemeproteins. Reaction (A) involves H₂S interaction with hemeproteins having nonpolar or hydrogen bond donor residues in their heme active site, while (B) engages the interaction of the bound gaseous ligand with hydrogen bond acceptor groups near the heme. Residues in E7, E11, and B10 positions are very important in regulating the binding and discrimination of ligands in hemeproteins.

FIG. 7.

Proposed mechanism for sulfheme formation. Suggested intermediates upon reaction of H₂O₂, in the presence of H₂S, with His distal Si proteins that produces the suflheme derivates.

In the context of the physiological function of HbI, we hypothesize that at low sulfide concentrations (1:1 stoichiometry), simple dissociation and heme reduction are involved in the delivery of H₂S to the symbiotic bacteria. However, under these conditions, the slow release of H₂S dominates over the heme reduction reaction. On the other hand, at high H₂S concentrations, its delivery is facilitated by the heme reduction reaction, which simultaneously promotes the formation of polysufides, and elemental sulfur. These elemental sulfur compounds are then stored within clam tissues and used by the bacterial endosymbionts during sulfide starvation or at low H₂S concentrations. Indeed, Lechaire and coworkers showed that the gill tissues of L. pectinata contain elemental sulfur produced by the clam (42). They suggested that this compound may act as an energy source for the bacteria during sporadic depletion of environmental H₂S.

In humans, H₂S might also be stored in the body as polysulfides and released in response to physiologic stimuli (Fig. 2) (34, 38). Therefore, heme reduction and polysulfide and elemental sulfur formation may be relevant products resulting from the interaction of H₂S with hemeproteins in our body. In fact, these products have been suggested to form in CcO and flavocytochrome c sulfide dehydrogenase (53–55, 73).

Hbs from the worm Riftia pachyptila

The giant tubeworm R. pachyptila is another interesting invertebrate that lives in the sulfide-rich deep-sea hydrothermal vents and is also characterized by the presence of symbiotic sulfide-oxidizing bacteria that need to be supplied with both H₂S and O₂ (2). Riftia supplies H₂S and O₂ to the endosymbionts by binding both ligands simultaneously at two different sites in its extracellular Hbs. Two of these Hbs are dissolved in the vascular blood of Riftia, and have been designated V1 and V2, and a third one, named C1, is found in the coelomic fluid of the worm (2, 26, 80). These Hbs are giant and complex proteins with molecular weights ranging from ∼ 3500 kDa for V1 to ∼400 kDa for V2 and C1. They appear to bind O₂ in their heme groups and H₂S in other sites remote from the hemes. It was initially thought that the binding of H₂S occurred at “free cysteine” or cysteine residues not involved in disulfide bonds (2, 80). However, the role of cysteines in H₂S binding was questioned by Flores and coworkers, who solved the crystal structure of the worm C1 Hb (26). They showed that this Hb (Fig. 8A) consists of 24 heme-containing globin chains tightly associated to form a hollow sphere with 12 zinc ions bound near the sphere. The authors showed that the Zn²⁺ ions bind H₂S readily and that the cysteine residues previously proposed as sulfide-binding sites were located in hydrophobic environments that might restrict access to H₂S.

FIG. 8.

Structure of Riftia pachyptila C1 Hb (PDB: 1YHU) and its reactivity with H₂S. (A–C) C1 Hb quaternary structure, heme active site, and its reaction with H₂S, respectively. The nomenclature in (B) refers to the positions of the amino acids near the heme.

Whether H₂S binds to the Zn²⁺ ions or the cysteine residues is still unclear; however, what is clear is that the heme groups in the Hbs from R. pachyptila do not interact directly with the gas. Instead, these sites are limited to O₂ binding only. The exclusion of H₂S from the heme cavity might be due to restricted accessibility. As Figure 8A shows, the protein is gigantic in comparison to the other Hbs (Figs. 5A and 9A), which likely plays a role in restraining rapid access of H₂S to the hemes as discussed below. In fact, it has been shown that H₂S binding to HbI from L. pectinata is nearly 1000 times slower than binding of O₂, suggesting that protein fluctuations are required to allow sulfide access to the heme distal site probably due to the bulkier character of H₂S (40, 61). Thus, direct binding of H₂S to the hemes in Riftia Hb may require large protein fluctuations, delaying in turn the entrance of the ligand to the distal active site. In addition, as shown in Figure 8A, the hemes (in red) are buried in the protein matrix, consistent with the model that H₂S binding to the zincs, which are located in the exposed hollow sphere, is more facile (26). The binding of H₂S to the zinc ion sites would have the benefit of avoiding sulfheme formation. Although the C1 Hb in Riftia has a histidine residue near the heme (Fig. 8B), formation of a sulfheme complex has not been detected. In contrast, sulfheme formation has been observed in Hbs from M. decora and L. terrestris, which live in sulfide-free environments and also contain histidine at the ligand binding site (67, 74, 78). Similar to Riftia, these Hbs are large, consisting of 144 heme-containing globin chains with an analogous quaternary structure. Structural studies have demonstrated that “free cysteine residues” are absent in these Hbs and that if zinc is present, the content is low (e.g., one zinc atom in Lumbricus hemoglobin) (2, 72). This implies that in the presence of O₂, H₂S diffuses through these proteins without encountering alternative binding sites and once at the heme site, can interact directly with heme forming the sulfheme derivatives. These observations also support the notion that histidine is required for the formation of the sulfheme derivatives. On this basis, we suggest that in R. pachyptila, the zinc ions (or the cysteine residues) transport H₂S to the symbiotic bacteria and protect the worm from H₂S toxicity by impeding access to the hemes, thus avoiding sulfheme formation, which can limit O₂ transportation.

FIG. 9.

Structure of Thermobifida fusca truncated Hb (PDB: 2BMM) and its reactivity with H₂S. (A–C) T. fusca trHb tertiary structure, heme active site, and its reaction with H₂S, respectively. The nomenclature in (B) refers to the positions of the amino acids near the heme.

Hbs from Bacillus subtilis and Thermobifida fusca

B. subtilis and T. fusca are rod-shaped bacteria that are usually found in soils and can tolerate extreme environmental conditions. These organisms have specialized truncated Hbs (Bs-trHb for B. subtilis and Tf-trHb for T. fusca) that have high affinity for H₂S and are believed to play a possible physiological role in the bacteria (56). In particular, these Hbs have been suggested to be involved in sulfur metabolism in these microorganisms, including cysteine biosynthesis and thiol-based redox homeostasis. While direct experimental evidence for the role of Hb in sulfur metabolism is lacking, the high affinity of both Bs-trHb (5.0 × 10⁶ M⁻¹) and Tf-trHb (2.8 × 10⁶ M⁻¹) for H₂S provides relevant paradigms for studying the interaction of H₂S with hemeproteins.

It has been recently shown that, like HbI from L. pectinata, the affinity of the trHbs for H₂S results from the very rapid association and extremely slow dissociation kinetics (Table 2) (56). Like HbI, these trHbs are monomeric proteins (Fig. 9A) and their H₂S binding constants suggest that the gas has rapid access to the bound heme. The crystal structure of Tf-trHb (Fig. 9B) reveals that its distal site is characterized by the presence of aromatic amino acids surrounding the heme group, including tyrosine residues and a tryptophan residue. As shown recently, replacement of tryptophan, which has a larger volume (227.8 ų) with phenylalanine, which has smaller volume (189.9 ų) (81), increases the association constant by a factor of 50, consistent with the suggestion that in the trHbs, H₂S association is controlled in part by the space in the vicinity of the heme (56). Moreover, since the rate of sulfide association is ∼2000 slower than of O₂ in Tf-trHb, protein fluctuations that allow access of the larger H₂S ligand to the distal site may also be required in this Hb (11, 27).

Table 2.

Oxygen and H₂S Association and Dissociation Constants of Lucina pectinata HbI, Bacillus subtilis, and Thermobifida fusca Truncated Hemoglobins and a Tf-TrpG8Phe Mutant

Protein	FeIII-H2S kon (M−1S−1)	FeIII-H2S koff (S−1)	FeII-O2 kon (M−1S−1)	FeII-O2 koff (S−1)
L. Pectinata Hbla	2.3 × 105	0.00022	100–200 × 106	61
Bs-trHb	1.3 × 104,b	0.0026b	30 × 106,c	0.0048c
Tf-trHb	5 × 103,b	0.0018b	0.9 × 106,d	0.07d
Tf-TrpG8Phe	4.1 × 104,b	0.36b

^aData from ref. (40).

^bData from ref. (56).

^cData from ref. (28).

^dData from ref. (11).

TrHb, truncated hemoglobins.

To explain the slow H₂S dissociation observed in these trHbs, it was suggested that tryptophan acts as a hydrogen bond donor in both Bs-trHb and Tf-trHb, stabilizing the bound sulfide, and retarding its release. Unlike HbI, this electrostatic interaction does not promote heme reduction and H₂S release. This does not however contradict the reaction scheme proposed in Figure 6. As stated above, heme reduction is only invoked for proteins having residues with hydrogen bond acceptor groups near the heme. From an electrostatic point of view, one can infer that when hydrogen bond donating residues interact with the bound H₂S, a significant charge transfer occurs from the lone pair electrons of the acceptor sulfur ligand to the orbital of the donor, thereby increasing the ionization potential of the gaseous ligand and consequently, its ability to donate an electron to the heme iron decreases (10).

Overall, the available structures of Hb C1 from R. pachyptila and the Hbs from B. subtilis, T. fusca, and L. pectinata, supports the model that binding of sulfide to hemeproteins is dictated by ligand accessibility to the heme group and the dielectric of the heme environment.

Role of H₂S Reactivity with Hemeproteins: Concluding Comments and Future Perspective

Based on the discussion of H₂S interactions with vertebrate and invertebrate hemeproteins, we can summarize that at least four reactions influence H₂S reactivity with hemeproteins: (i) binding of H₂S to alternate sites such as cysteine, copper, and zinc ions, (ii) coordination of H₂S to the ferric heme iron without inducing reduction or sulfheme production, (iii) binding of H₂S to the ferric iron with subsequent reduction of the heme, and (iv) incorporation of H₂S into one of the pyrrole rings of the heme, generating the sulfheme derivative. In hemeproteins having nonpolar or hydrogen bond donating residues in their heme binding site, H₂S is slowly liberated from the ferric heme without involvement of redox chemistry. In contrast, proteins with hydrogen bond acceptor groups near the heme, promote reduction of the heme iron by a second H₂S molecule with the concomitant formation of polysulfides and/or S°. Subsequent to heme reduction, hemeproteins having histidine residues in their active site can also form the sulfheme complex in the presence of O₂ and a slight H₂S excess (Fig. 7).

In human Hb and Mb as well as in catalase (52) and lactoperoxidase (49), the histidine residue near the heme might plays an essential role in sulfheme formation. In CcO, the histidine residues within the catalytic center do not have the proper orientation to interact directly with the heme a₃-O₂ moiety, preventing interaction with the heme-peroxo or oxo-ferryl intermediates, thus avoiding in turn sulfheme production. This might explain in part why H₂S induces reduction of the Cu_B and the heme a₃ center, in CcO without stimulating sulfheme formation. H₂S also reduces the heme iron of cytochrome c (18, 55). In cytochrome c, the heme is coordinated by a methionine and a histidine residue at the distal and proximal sites, respectively. Although the mechanism by which H₂S reduces the heme center in cytochrome c is unclear, it has been suggested that sulfide can bind and reduce the heme iron thus affecting the redox state of the protein (28, 55). Interestingly, the heme in CBS, the hemeprotein that catalyzes H₂S biosynthesis, also has a cysteine ligand at the distal site and a histidine at the proximal site (69, 70). Thus, the interaction of H₂S with CBS and cytochrome c warrants further investigations. Moreover, it has been suggested recently that human neuroglobin, a member the globin super-family, which is found in the central nervous system, might influence H₂S levels, thus protecting cells from sulfide toxicity (12). However, H₂S binding to neuroglobin appears to be complex and a thorough investigation of the mechanism of its interaction with sulfide is needed.

Based on the published literature, the reactivity of H₂S with hemeproteins can be summarized by the generalized reactions shown in Figures 6 and 7. Key issues that remain to be investigated include (a) the role of the distal heme a₃ environment in CcO reduction at low H₂S concentrations and the reduction of the Cu_B and the heme a₃ center at moderate concentrations, (b) the direct role of histidine in sulfheme formation, (c) the intermediates associated with sulfheme formation, (d) the reactivity of the H₂S/HS⁻ pair with other hemeproteins like cytochrome c, CBS, and human neuroglobin, and (e) identifying additional hemeprotein targets of H₂S. Insights into these issues will allow unraveling of the mysteries associated with H₂S reactivity in hemeproteins, and allow assessment of the physiological implications of these interactions.

Abbreviations Used

3MST

3 mercaptopyruvate sulfur transferase

CBS

cystathionine β-synthase

CcO

cytochrome c oxidase

CSE

cystathionine γ-lyase

Fe^II

deoxy heme

Fe^II-O₂

oxy heme

Fe^III-O₂^-

peroxo

Fe^III-OH₂

met heme

Fe^IV

O Por^∙+ = ferryl Compound I

Fe^IV

O = ferryl Compound II

Hb

hemoglobin

H₂S

hydrogen sulfide

K_ATP

ATP-sensitive potassium

Mb

myoglobin

PLP

pyridoxal phosphate

Sulfheme

sulfhemoglobin or sulfmyoglobin

trHb

truncated hemoglobins

Acknowledgments

This work was supported in part by funds from the National Science Foundation (Grant 0843608) and NIH-NIGMS/MBRS-SCORE 5 S06GM008103-36. We thank the graduate student Laura B. Granell for her assistance during the work.