Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria—the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.


Introduction
For a long time, hydrogen sulfide (H 2 S) had been considered merely as a highly toxic and occasionally lethal gas. However, it was discovered that in mammals H 2 S has physiological relevance and is endogenously generated [1,2]. Currently, H 2 S is considered to be a member of the class of gasotransmitters or, in other words, endogenous gaseous signaling molecules, along with nitric oxide and carbon monoxide [3][4][5][6][7]. Although still debated, cyanide has also recently been proposed to be part of this class [7,8]. H 2 S contributes to the regulation of important physiological processes in the cardiovascular, gastrointestinal, nervous, and respiratory systems. It shows various physiological effects in mammalian cells, but in a biphasic, concentration-dependent manner. At low, nanomolar concentrations, H 2 S exhibits cytoprotective effects. At higher levels the compound induces cytotoxicity. For instance, H 2 S exerts vasorelaxant effects through the opening of K ATP (adenosine triphosphate) channels in vascular smooth muscle cells [9]. It also functions as a neuromodulator in the brain [1] and serves as a stimulator of angiogenesis [10]. At concentrations of 200 µM and higher, H 2 S induces apoptosis of aorta smooth muscle cells through the activation of mitogen-activated protein kinases and caspase-3 [11]. In mammalian systems, H 2 S is The physiological roles of H2S are not limited to mammals but appear to be i in all kingdoms of life. In higher plants, H2S signaling functions seem to occur through a persulfidation-based mechanism [14]. It regulates important physi functions, including fruit ripening; stomatal movement; senescence in flowers, leav fruits; photosynthesis via promotion of photosynthetic enzyme expression and plast biogenesis; and the promotion of root organogenesis, seed germination, nod and N2 fixation. H2S can also activate antioxidant systems in plant cells, thereby uting to defense against adverse environment situations, such as drought-induced tive stress, salinity stress, temperature stress (high and low temperatures), toxi metal stress, as well as different biotic stresses (see [14] and references therein).
Although most bacteria can generate and sense H2S, the exact physiological this compound in the prokaryotes are not yet wholly understood. In particular, w endogenous H2S is a true signaling effector molecule that induces a response in th microbial cell, or whether the H2S-mediated effect is more a response to environ and/or host-derived H2S, remains to be established [6]. Meanwhile, Shatalin et al. r that inhibition of H2S production through inactivation of H2S-generating enzyme cillus anthracis Sterne, Pseudomonas aeruginosa PA14, Staphylococcus aureus (MSSA and MRSA MW2), and Escherichia coli MG1655 makes these pathogenic species v sitive to different classes of antibiotics [15]. The addition of exogenous H2S rever effect. The authors concluded that endogenously generated H2S increases bacte sistance to oxidative stress imposed by antibiotics. The antibiotic-induced RO DNA damage via the Fenton reaction. It is suggested that H2S prevents oxidativ damage in bacteria via the following cytoprotective mechanisms: (i) direct redu H2O2 into H2O; (ii) depletion of Fe 2+ , a catalyst of the Fenton reaction; (iii) transien tion of free cysteine, a reducing agent that fuels the Fenton reaction; and (iv) stim of the activities of superoxide dismutase (SOD) and catalase [15]. The latter two e are the well-known ROS scavenging systems [16]. It should be noted, however, th The physiological roles of H 2 S are not limited to mammals but appear to be inherent in all kingdoms of life. In higher plants, H 2 S signaling functions seem to occur mainly through a persulfidation-based mechanism [14]. It regulates important physiological functions, including fruit ripening; stomatal movement; senescence in flowers, leaves, and fruits; photosynthesis via promotion of photosynthetic enzyme expression and chloroplast biogenesis; and the promotion of root organogenesis, seed germination, nodulation, and N 2 fixation. H 2 S can also activate antioxidant systems in plant cells, thereby contributing to defense against adverse environment situations, such as drought-induced oxidative stress, salinity stress, temperature stress (high and low temperatures), toxic heavy metal stress, as well as different biotic stresses (see [14] and references therein).
Although most bacteria can generate and sense H 2 S, the exact physiological roles of this compound in the prokaryotes are not yet wholly understood. In particular, whether endogenous H 2 S is a true signaling effector molecule that induces a response in the same microbial cell, or whether the H 2 S-mediated effect is more a response to environmental and/or host-derived H 2 S, remains to be established [6]. Meanwhile, Shatalin et al. reported that inhibition of H 2 S production through inactivation of H 2 S-generating enzymes in Bacillus anthracis Sterne, Pseudomonas aeruginosa PA14, Staphylococcus aureus (MSSA RN4220 and MRSA MW2), and Escherichia coli MG1655 makes these pathogenic species very sensitive to different classes of antibiotics [15]. The addition of exogenous H 2 S reverses this effect. The authors concluded that endogenously generated H 2 S increases bacterial resistance to oxidative stress imposed by antibiotics. The antibiotic-induced ROS cause DNA damage via the Fenton reaction. It is suggested that H 2 S prevents oxidative DNA damage in bacteria via the following cytoprotective mechanisms: (i) direct reduction of H 2 O 2 into H 2 O; (ii) depletion of Fe 2+ , a catalyst of the Fenton reaction; (iii) transient depletion of free cysteine, a reducing agent that fuels the Fenton reaction; and (iv) stimulation of the activities of superoxide dismutase (SOD) and catalase [15]. The latter two enzymes are the well-known ROS scavenging systems [16]. It should be noted, however, that Shatalin et al. [15] did not suggest a mechanism for the depletion of Fe 2+ and free cysteine by H 2 S. H 2 S is a colorless flammable gas, soluble in water (100 mM at 25 • C [17]), and with high cell membrane permeability [18]. It is a weak acid; therefore in aqueous solutions, it equilibrates with hydrosulfide (HS − ) and sulfide (S 2− ) according to Equation (1): According to the reported pK a1 values that varied from 6.97 to 7.06 at 25 • C (see [19] and references therein), at physiological pH 7.4, 69-73% of the total hydrogen sulfide pool in the solution is in the form of HS − , and 27-31% exists as H 2 S. Taking into account the reported pK a2 values (from 12.2 to 19), the concentration of S 2− in the solution at pH 7.4 is negligible. The mitochondrial matrix in eukaryotic cells is usually alkaline, with a pH value of about 8 [20,21]. Accordingly, in the matrix, the concentration of HS − is higher, 90-91%, and the remainder is in the form of H 2 S. On the contrary, intralysosomal pH in living cells is highly acidic, 4.7-4.8 [22]. Therefore, inside lysosomes, the undissociated form of hydrogen sulfide, H 2 S, dominates (>99%). H 2 S acts as a reducing agent. Respectively, the standard redox potential (vs. the standard hydrogen electrode, pH 7) E 0 (S 0 /H 2 S) is −230 mV (E 0 (S 0 /HS − ) = −270 mV) [19]. Herein, unless otherwise stated, we use the term "H 2 S" to designate the total hydrogen sulfide pool (H 2 S + HS − + S 2− ).
This review focuses on the effects of H 2 S on the respiratory chains of mammalian mitochondria and bacteria, mammalian F o F 1 -ATP synthase, and mammalian lactate dehydrogenase (LDH) in light of recent findings.

Endogenous Production of H 2 S
In mammalian tissues, H 2 S can be endogenously produced via both non-enzymatic and enzymatic pathways.

Non-Enzymatic Production of H 2 S
Non-enzymatic formation of H 2 S usually takes place in the reactions of thiols or thiol derivatives with other molecules [12,13,23]. Inorganic polysulfides, persulfides, and thiosulfate can be reduced with reduced glutathione (GSH) to yield H 2 S ( Figure 2). This demands the presence of reducing equivalents, such as NADPH (reduced nicotinamide adenine dinucleotide phosphate), because glutathione disulfide (GSSG), also produced in the reaction, needs to be converted back to GSH by NADPH-dependent glutathione reductase. Inorganic sulfide salts, such as Na 2 S or NaHS, can undergo hydrolysis. Yang et al. also reported that cysteine is the preferred substrate for the non-enzymatic pathway, and that the reaction required coordinated catalysis by Fe 3+ and pyridoxal phosphate (PLP) [24] (Figure 2).
H2S is a colorless flammable gas, soluble in water (100 mM at 25 °C [17]), and high cell membrane permeability [18]. It is a weak acid; therefore in aqueous solutio equilibrates with hydrosulfide (HS − ) and sulfide (S 2− ) according to Equation (1): According to the reported pKa1 values that varied from 6.97 to 7.06 at 25 °C (see and references therein), at physiological pH 7.4, 69-73% of the total hydrogen sulfide in the solution is in the form of HS − , and 27-31% exists as H2S. Taking into accoun reported pKa2 values (from 12.2 to 19), the concentration of S 2− in the solution at pH 7 negligible. The mitochondrial matrix in eukaryotic cells is usually alkaline, with a value of about 8 [20,21]. Accordingly, in the matrix, the concentration of HS − is higher 91%, and the remainder is in the form of H2S. On the contrary, intralysosomal pH in li cells is highly acidic, 4.7-4.8 [22]. Therefore, inside lysosomes, the undissociated for hydrogen sulfide, H2S, dominates (>99%). H2S acts as a reducing agent. Respectively standard redox potential (vs. the standard hydrogen electrode, pH 7) E 0 ′(S 0 /H2S) is mV (E 0 ′(S 0 /HS -) = −270 mV) [19]. Herein, unless otherwise stated, we use the term " to designate the total hydrogen sulfide pool (H2S + HS − + S 2− ).
This review focuses on the effects of H2S on the respiratory chains of mamm mitochondria and bacteria, mammalian FoF1-ATP synthase, and mammalian lactate d drogenase (LDH) in light of recent findings.

Endogenous Production of H2S
In mammalian tissues, H2S can be endogenously produced via both non-enzym and enzymatic pathways.

Non-Enzymatic Production of H2S
Non-enzymatic formation of H2S usually takes place in the reactions of thiols or derivatives with other molecules [12,13,23]. Inorganic polysulfides, persulfides, and sulfate can be reduced with reduced glutathione (GSH) to yield H2S ( Figure 2). Thi mands the presence of reducing equivalents, such as NADPH (reduced nicotinamid enine dinucleotide phosphate), because glutathione disulfide (GSSG), also produce the reaction, needs to be converted back to GSH by NADPH-dependent glutathion ductase. Inorganic sulfide salts, such as Na2S or NaHS, can undergo hydrolysis. Yan al. also reported that cysteine is the preferred substrate for the non-enzymatic path and that the reaction required coordinated catalysis by Fe 3+ and pyridoxal phosp (PLP) [24] (Figure 2).

Enzymatic Production of H 2 S
Enzymatic production of H 2 S in mammalian systems is carried out primarily by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvatesulfurtransferase (3MST) [12,13,23,25,26]. The main enzymatic reactions resulting in the biosynthesis of H 2 S are shown in Figure 3. CBS can catalyze the condensation of homocysteine and L-cysteine to produce L-cystathionine and H 2 S (Figure 3, reaction 1). In the presence of L-cysteine, CBS can also produce H 2 S, with the formation of L-serine as a byproduct ( Figure 3, reaction 2). CSE can decompose homocysteine to yield H 2 S, α-ketobutyrate, and ammonia ( Figure 3, reaction 3). Furthermore, CSE can catalyze the conversion of L-cysteine into H 2 S, pyruvate, and ammonia ( Figure 3, reaction 4). Both CBS and CSE can generate H 2 S through β-replacement of cysteine by a second cysteine, with the formation of lanthionine as a byproduct ( Figure 3, reaction 5). Additionally, CSE can catalyze the γ-replacement reaction between two homocysteine molecules, with the production of H 2 S and homolanthionine as a by-product (Figure 3, reaction 6). 3MST generates H 2 S from 3-mercaptopyruvate coupled with either of the two enzymes, mitochondrial cysteine aminotransferase (CAT) or peroxisomal D-amino acid oxidase (DAO). 3MST transfers a sulfur atom from 3-mercaptopyruvate onto itself, resulting in the formation of the enzyme-bound persulfide (3MST-SS) and pyruvate ( Figure 3, reaction 7). H 2 S is then released from the persulfide in the presence of a reductant, e.g., thioredoxin (Trx). CAT and DAO in turn produce 3-mercaptopyruvate from L-cysteine and D-cysteine, respectively [13].
Int. J. Mol. Sci. 2021, 22, x 4 sulfurtransferase (3MST) [12,13,23,25,26]. The main enzymatic reactions resulting in biosynthesis of H2S are shown in Figure 3. CBS can catalyze the condensation of ho cysteine and L-cysteine to produce L-cystathionine and H2S ( Figure 3, reaction 1). In presence of L-cysteine, CBS can also produce H2S, with the formation of L-serine as a product ( Figure 3, reaction 2). CSE can decompose homocysteine to yield H2S, α-keto tyrate, and ammonia ( Figure 3, reaction 3). Furthermore, CSE can catalyze the conver of L-cysteine into H2S, pyruvate, and ammonia ( Figure 3, reaction 4). Both CBS and can generate H2S through β-replacement of cysteine by a second cysteine, with the mation of lanthionine as a byproduct ( Figure 3, reaction 5). Additionally, CSE can cata the γ-replacement reaction between two homocysteine molecules, with the productio H2S and homolanthionine as a by-product (Figure 3, reaction 6). 3MST generates H2S f 3-mercaptopyruvate coupled with either of the two enzymes, mitochondrial cysteine a notransferase (CAT) or peroxisomal D-amino acid oxidase (DAO). 3MST transfers a su atom from 3-mercaptopyruvate onto itself, resulting in the formation of the enzy bound persulfide (3MST-SS) and pyruvate ( Figure 3, reaction 7). H2S is then released f the persulfide in the presence of a reductant, e.g., thioredoxin (Trx). CAT and DAO in produce 3-mercaptopyruvate from L-cysteine and D-cysteine, respectively [13]. The majority of bacterial species whose genomes were completely sequenced h the orthologs of mammalian genes encoding CBS, CSE, or 3MST [15]. Since H2S prov defense against modern antibiotics in bacteria, suppression of H2S-producing enzyme pathogens by new drugs would be a promising antimicrobial treatment strategy [15 The use of H2S biogenesis as a target for versatile antibiotic potentiators may have th peutic potential for the fight against difficult-to-treat infections based on bacterial an otic tolerance.

S-Sulfhydration of ATP Synthase
In eukaryotes, one of the main targets of H2S signaling is the mitochondria. These organelles are known to be the power plants of the eukaryotic cell. Energy transduc The majority of bacterial species whose genomes were completely sequenced have the orthologs of mammalian genes encoding CBS, CSE, or 3MST [15]. Since H 2 S provides defense against modern antibiotics in bacteria, suppression of H 2 S-producing enzymes in pathogens by new drugs would be a promising antimicrobial treatment strategy [15,27]. The use of H 2 S biogenesis as a target for versatile antibiotic potentiators may have therapeutic potential for the fight against difficult-to-treat infections based on bacterial antibiotic tolerance.

S-Sulfhydration of ATP Synthase
In eukaryotes, one of the main targets of H 2 S signaling is the mitochondria. These cell organelles are known to be the power plants of the eukaryotic cell. Energy transduction events in mitochondria occur in the O 2 -dependent respiratory electron transport chain. The mammalian respiratory chain is unbranched [28,29]. It consists of four membranebound multi-subunit complexes: I, (reduced nicotinamide adenine dinucleotide) NADH: ubiquinone reductase or type I NADH dehydrogenase; II, succinate dehydrogenase; III, ubiquinol: cytochrome c reductase or cytochrome bc 1 complex; and IV, cytochrome c oxidase or cytochrome aa 3 . The chain transfers electrons from NADH and succinate to O 2 . The electron transfer reactions catalyzed by complexes I, III, and IV are coupled to the generation of the proton motive force. The latter is used by ATP synthase (F o F 1 -ATP synthase or complex V) to produce ATP.
Modis et al. observed S-sulfhydration of subunit α of the synthase (ATP5A1) in HepG2 and HEK293 cell lysates in response to exposure to H 2 S, using a biotin switch assay [30]. Sulfhydration of subunit α of the synthase increases with increasing the H 2 S concentration, 50-300 µM. H 2 S at low concentrations (10-100 nM) stimulates the specific activity of ATP synthase, while at higher concentrations (1-10 µM) a tendency for inhibition of the activity is detected (Figure 4). Such a bell-shaped concentration-response curve is quite typical for the effects of H 2 S. Sulfhydration occurs at two highly conserved cysteine residues in subunit α of the synthase, Cys244, or Cys294. Mutation of either of the two cysteines (C244S or C294S) leads to a slight reduction in the catalytic activity of ATP synthase. The double mutant (C244S/C294S) exhibits more than 50% inhibition of the enzyme activity. In vivo, subunit α of the synthase is basally sulfhydrated. The basal sulfhydration is mostly due to CSE-derived endogenous H 2 S generation because it is suppressed in liver homogenates harvested from CSE −/− mice. Burn injuries that upregulate CSE and increase H 2 S generation result in an increase in S-sulfhydration of subunit α of the synthase. Thus, S-sulfhydration of subunit α of the synthase could be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics [30]. mostly due to CSE-derived endogenous H2S generation because it is suppressed in liver homogenates harvested from CSE −/− mice. Burn injuries that upregulate CSE and increase H2S generation result in an increase in S-sulfhydration of subunit α of the synthase. Thus, S-sulfhydration of subunit α of the synthase could be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics [30]. The mammalian respiratory chain includes four different membrane-bound complexes: I, II, III, and IV. H2S at low concentrations stimulates the activity of FoF1-ATP synthase (also known as complex V) via S-sulfhydration of Cys244 or Cys294 of its α subunit [30]. H2S increases the activity of lactate dehydrogenase (LDH) via S-sulfhydration of its Cys163 that, in turn, stimulates mitochondrial electron transport [31]. H2S can also donate electrons to the respiratory chain via sulfide quinone oxidoreductase (SQOR) by directly reducing ubiquinone (UQ) [32]. At high (toxic) concentrations H2S inhibits complex IV (cytochrome c oxidase) and FoF1-ATP synthase that leads to reversible inhibition of mitochondrial electron transport and ATP production [30,[33][34][35]. At low concentrations H2S serves as an electron donor for complex IV, Figure 4. Effects of H 2 S on mammalian mitochondrial electron transport chain, ATP synthase, and lactate dehydrogenase. The mammalian respiratory chain includes four different membrane-bound complexes: I, II, III, and IV. H 2 S at low concentrations stimulates the activity of F o F 1 -ATP synthase (also known as complex V) via S-sulfhydration of Cys244 or Cys294 of its α subunit [30]. H 2 S increases the activity of lactate dehydrogenase (LDH) via S-sulfhydration of its Cys163 that, in turn, stimulates mitochondrial electron transport [31]. H 2 S can also donate electrons to the respiratory chain via sulfide quinone oxidoreductase (SQOR) by directly reducing ubiquinone (UQ) [32]. At high (toxic) concentrations H 2 S inhibits complex IV (cytochrome c oxidase) and F o F 1 -ATP synthase that leads to reversible inhibition of mitochondrial electron transport and ATP production [30,[33][34][35]. At low concentrations H 2 S serves as an electron donor for complex IV, either directly [36][37][38] or indirectly, via reduction of its native substrate cytochrome c [39]. The outer mitochondrial membrane is not shown for simplicity.

S-Sulfhydration of Lactate Dehydrogenase (LDH)
LDH catalyzes the reversible conversion of lactate to pyruvate with the reduction of NAD + to NADH, and vice versa. The enzyme is a tetramer that is usually composed of the two most common types of subunits, LDHA and LDHB [40]. LDHA and LDHB can assemble into five different isoenzymes: LDH1, LDH2, LDH3, LDH4, and LDH5. Isoenzymes that are rich in LDHA catabolize pyruvate to lactate with the concomitant production of NAD + from NADH. Conversely, isoenzymes rich in LDHB facilitate lactateto-pyruvate conversion with the concomitant formation of NADH from NAD + .
Untereiner et al. reported that in the colon cancer cell line HCT116, LDHA catalyzes the conversion of pyruvate to lactate, and H 2 S donation increases the total cellular lactate levels [31]. Notably, H 2 S enhances the catalytic activity of LDHA, which is primary cytosolic, doing so via S-sulfhydration of its Cys163 (Figure 4). Experiments with whole HCT116 cell extracts showed that H 2 S also stimulates the LDHB activity, although to a smaller extent. Importantly, H 2 S stimulates oxidative phosphorylation in HCT116 cells in an LDHA-dependent manner. Thus, in colon cancer cells, H 2 S-induced stimulation of the catalytic activity of LDHA leads to the stimulation of mitochondrial electron transport.
The authors hypothesize that the increase in the LDHA activity causes an increase in cytosolic lactate. This enhances the flux of lactate into the mitochondria through the intracellular lactate shuttle. Lactate enters the mitochondrial intermembrane space. Lactate and pyruvate also enter the mitochondrial matrix. Lactate is converted to pyruvate via the mitochondrial LDH that is primarily made up of LDHB and also activated by H 2 S. Pyruvate in the matrix is oxidized via the Krebs cycle to generate NADH that stimulates the activity of the mitochondrial respiratory chain. Additionally, the oxidation of lactate to pyruvate by the mitochondrial LDH is coupled to the reduction of NAD + to NADH. This NADH, in turn, is utilized by the mitochondrial respiratory chain to further support electron transport.

H 2 S Donates Electrons to the Mitochondrial Respiratory Chain via Sulfide Quinone Oxidoreductase (SQOR)
Powell and Somero first reported that the oxidation of H 2 S can occur in mitochondria, and that the process is coupled to oxidative phosphorylation [41]. The mitochondrial oxidation of H 2 S was observed in the gill and foot tissue of Solemya reidi, a gutless clam living in sulfide-rich habitats. Later, Goubern et al., using permeabilized human colon adenocarcinoma HT29 cells, showed that H 2 S at low micromolar concentrations can serve as an electron donor for the mammalian respiratory chain [32]. This is accompanied by mitochondrial energization. The latter is extremely sensitive to the amount of H 2 S delivered instantaneously to mitochondria. H 2 S donates electrons at the respiratory chain at the level of UQ.
The enzyme that catalyzes the electron transfer from H 2 S to UQ is SQOR [42]. SQOR belongs to group four of the flavin disulfide reductase (FDR) superfamily. The enzyme contains a tightly bound FAD (flavin adenine dinucleotide) and two spatially proximal redox-active cysteines. A peculiar feature of human SQOR is the fact that the two cysteines are linked via a bridging sulfur atom which forms the redox-active cysteine trisulfide configuration. The trisulfide appears to be the active form of SQOR, being present at the start and end of the catalytic cycle [43].
When H 2 S is oxidized in the mitochondrial matrix by the action of SQOR under physiological conditions, GSH serves as the primary sulfur acceptor (Figure 4). In the reaction that is coupled to the reduction of UQ to UQH 2 , glutathione persulfide (GSSH) is produced. GSSH is then oxidized by persulfide dioxygenase (ETHE1) to yield sulfite (SO 3 2− ) and regenerate GSH. GSSH can also be a substrate for rhodanese or thiosulfate sulfurtransferase (TST). TST catalyzes the reaction of GSSH with SO 3 2− to generate GSH and thiosulfate (S 2 O 3 2− ). Alternatively, SO 3 2− can be oxidized to sulfate (SO 4 2− ) by sulfite oxidase (SUOX) located in the intermembrane space, with the concomitant reduction of cytochrome c. Thiosulfate and sulfate can be excreted with urine (see [42] and references therein). Thus, electrons from the sulfide oxidation pathway can enter the mitochondrial respiratory chain at the level of the cytochrome bc 1 complex (from SQOR) and cytochrome c oxidase (from SUOX).

H 2 S at Toxic Concentrations Inhibits Mitochondrial Cytochrome c Oxidase
H 2 S exposure, at high concentrations and high rates, is extremely toxic to mammals, including humans [35]. The acute toxicity of H 2 S is generally attributed to the suppression of the mitochondrial cytochrome c oxidase (complex IV). Complex IV couples the transfer of electrons from cytochrome c to O 2 with the generation of the transmembrane proton gradient using the mechanism of proton pumping [16,. The enzyme carries four redox-active metal sites: Cu A , heme a, heme a 3 , and Cu B . Electrons from cytochrome c primarily accepted by Cu A are transferred to heme a and then to the catalytic binuclear center composed of heme a 3 and Cu B in which the reduction of oxygen to water occurs [66][67][68][69]. There are two forms of cytochrome c oxidase: slow (resting oxidase as obtained from the preparation) and fast (the completely reduced enzyme exposed to a "pulse" of O 2 ) [70]. The fast form is also called pulsed or unrelaxed. The slow form differs from the fast form in lower reactivity for inhibitors [71] and slower intramolecular electron transfer [72].
H 2 S at high concentrations was reported to inhibit the O 2 consumption by the beef heart cytochrome c oxidase in the isolated form and in submitochondrial particles ( Figure 4) [33,34,73]. The inhibition by H 2 S appears to be non-competitive with respect to both substrates, cytochrome c and O 2 [34]. The inhibition efficiency increases with decreasing pH: the K i values measured at pH 8.05, 7.48, and 6.28, are 2.6, 0.55, and 0.07 µM, respectively [37]. The initial rate of inactivation of the fast form of the isolated cytochrome c oxidase is proportional to total H 2 S concentration, with an initial rate constant, k on , of 2.2 × 10 4 M −1 s −1 at pH 7.47 [38]. The inhibition of complex IV leads to blockage of the mitochondrial respiratory chain and, as a consequence, to the dissipation of the membrane potential, the cessation of oxidative phosphorylation, and enhanced ROS production [35]. Consistently, examination of the cytotoxic effects of inhalational H 2 S exposure showed that sublethal (>50 ppm) concentrations of the inhaled gas caused inhibition of cytochrome c oxidase activity in the lung and heart, which can be observed ex vivo and is associated with pathological alterations [74][75][76][77][78][79]. That complex IV is suppressed by high (usually 10-100 µM) concentrations of H 2 S in vitro in various cell lines is well documented [75,[80][81][82][83][84][85]. The H 2 S-induced inhibition is reversible, e.g., 10-30 min after the exposure of tissue homogenates to H 2 S, the activity of complex IV gets back to its initial level, simultaneously with the decomposition of the inhibitor [86].
H 2 S, presumably in the form of HS − , binds to the ferric heme a 3 and Cu B in either state (either cupric or cuprous) [38]. The final enzyme product of H 2 S inhibition is a mixed-valence state of the oxidase in which Cu A and heme a are reduced and heme a 3 is in the ferric H 2 S-bound form [33,87]. Cu B in the final inhibited enzyme remains reduced even after reoxidation of the H 2 S-bound oxidase, as evidenced by an electron paramagnetic resonance spectroscopy study [88]. The fact that Cu B remains cuprous in the presence of ferricyanide suggests that H 2 S binding to Cu B 1+ increases its redox potential and makes re-oxidation difficult. In other words, Cu B 1+ in this state may also be H 2 S-bound [88]. Nicholls et al. [38] suggested a model for the H 2 S-mediated inhibition of the mitochondrial cytochrome c oxidase ( Figure 5). According to this model, during the steady-state turnover of complex IV, the first molecule of HS − transiently binds to cupric or cuprous Cu B . Then, it is transferred to the ferric heme a 3 that blocks the catalytic reaction of the heme with O 2 . In the final protein-inhibitor adduct, Cu B is likely reduced and presumably bound to the second molecule of HS − . Heme a and Cu A are most likely reduced in the adduct.

H2S at Low Concentrations Serves as Electron Donor for Mitochondrial Cytochr c Oxidase
H2S at low concentrations can act as a substrate for mitochondrial complex IV (F 4) [36,37]. The fast form of the oxidized cytochrome c oxidase reacts aerobically with H2S levels in the absence of reduced substrates. However, the initial product of this tion is not the inhibited enzyme but a catalytic intermediate, compound 'P' (Figure 6)

H 2 S at Low Concentrations Serves as Electron Donor for Mitochondrial Cytochrome c Oxidase
H 2 S at low concentrations can act as a substrate for mitochondrial complex IV ( Figure 4) [36,37]. The fast form of the oxidized cytochrome c oxidase reacts aerobically with low H 2 S levels in the absence of reduced substrates. However, the initial product of this reaction is not the inhibited enzyme but a catalytic intermediate, compound 'P' (Figure 6) [38]. Compound 'P' then decays into another catalytic intermediate, compound 'F'. It is proposed that in this reaction two H 2 S molecules donate two electrons to the fully oxidized binuclear center, a 3 3+ Cu B 2+ , converting it to the fully reduced state, a 3 2+ Cu B + . The concomitant H 2 S oxidation product is possibly hydrogen persulfide (HSSH), although a form of free sulfur (S 0 or S 8 ) cannot be excluded [38]. Then the fully reduced binuclear center reacts with O 2 , producing compound 'P'. It remains unclear whether cytochrome c oxidase contributes significantly to a dissimilatory mechanism for the endogenously generated H 2 S, or if SQOR is the major contributor in vivo. Notably, H 2 S can also reduce complex IV indirectly, via reduction of its native substrate cytochrome c (Figure 4). The reaction between H 2 S and cytochrome c presumably leads to the initial formation of the HS • /S •− radical. HS • /S •− could then be trapped by proteins producing protein persulfides and superoxide, or be trapped by O 2 yielding HSO 2 states. The center consists of the copper ion CuB and the high-spin heme (Fehs). The latter in cytochrome c oxidase and heme o3 in cytochrome bo3. The metal redox groups which ar of the center are not shown.

H2S at Low Concentrations Serves as Electron Donor for Mitochondrial Cyto c Oxidase
H2S at low concentrations can act as a substrate for mitochondrial complex IV 4) [36,37]. The fast form of the oxidized cytochrome c oxidase reacts aerobically H2S levels in the absence of reduced substrates. However, the initial product of tion is not the inhibited enzyme but a catalytic intermediate, compound 'P' (Figur Compound 'P' then decays into another catalytic intermediate, compound 'F'. posed that in this reaction two H2S molecules donate two electrons to the fully binuclear center, a3 3+ CuB 2+ , converting it to the fully reduced state, a3 2+ CuB + . The c tant H2S oxidation product is possibly hydrogen persulfide (HSSH), although a free sulfur (S 0 or S8) cannot be excluded [38]. Then the fully reduced binuclear cen with O2, producing compound 'P'. It remains unclear whether cytochrome c oxid tributes significantly to a dissimilatory mechanism for the endogenously genera or if SQOR is the major contributor in vivo. Notably, H2S can also reduce compl directly, via reduction of its native substrate cytochrome c (Figure 4). The reaction H2S and cytochrome c presumably leads to the initial formation of the HS • /S • HS • /S •− could then be trapped by proteins producing protein persulfides and sup or be trapped by O2 yielding HSO2 • [39].

Effect of H 2 S on the Operation of the Branched Respiratory Chain of E. coli and Bacterial Growth
Under steady-state conditions, the mammalian tissue concentration of H 2 S is usually in the low nanomolar range, e.g., it is around 15 nM in mouse brain and liver tissues [89]. The mammalian gut, in this sense, is unique among other body compartments. Millimolar concentrations (1.0-2.4 mM) of H 2 S are commonly present in the gut [90]. The reason is that in the gastrointestinal tract, unlike other compartments, H 2 S is generated by both the mammalian CBS, CSE, and 3MST, and by the microbial communities inhabiting the gut, of which sulfate-reducing bacteria are the key H 2 S-producing species [90,91]. At such extremely high H 2 S levels, bacterial respiratory chains which terminate in cytochrome c oxidase or other heme-copper oxidases should be inhibited. The question then arises as to how under these conditions bacteria inhabiting the mammalian gut can maintain aerobic respiration. It turned out that E. coli, and possibly other bacteria inhabiting H 2 S-rich environments, has a unique bd-type terminal oxidase that is not inhibited by H 2 S, even at toxic concentrations [92][93][94][95].
Forte et al. studied the effect of H 2 S on the oxygen consumption by the purified terminal oxidases bo 3 , bd-I, and bd-II from E. coli [92]. It turned out that the activity of cytochrome bo 3 is quickly inhibited with an apparent half-maximal inhibitory concentration IC 50 of 1.1 µM H 2 S (pH 7.4). This value is similar to the K i of 0.55 µM H 2 S obtained by Nicholls and Kim for the beef heart cytochrome c oxidase at pH 7.48 [37]. The inhibition of the bo 3 oxidase appeared to be fully reversible. The rapid and total recovery of the enzymatic activity occurs following the removal of H 2 S from the solution by the H 2 S scavenger, the Entamoeba histolytica O-acetylserine sulfhydrylase (EhOASS), in the presence of excess O-acetyl-L-serine (OAS). In contrast, neither the bd-I oxidase nor the bd-II oxidase is inhibited under identical conditions, even at a high concentration of H 2 S (58 µM) [92]. Similar results were obtained when examining the effect of H 2 S on the oxygen consumption by cell suspensions of the E. coli mutant strains which possess bo 3 , bd-I, and bd-II as the only terminal oxidase. The oxygen uptake by cells having bo 3 as the sole oxidase is rapidly inhibited by 50 µM H 2 S. As in the case of the purified enzyme, the inhibition is promptly and completely restored after H 2 S depletion by the EhOASS/OAS system. Conversely, H 2 S at the same concentration does not affect the oxygen uptake by mutant cells having either bd-I or bd-II as the only oxidase [92]. Similar data on the E. coli membrane vesicles were reported by Korshunov et al. [93]. Forte et al. studied the effect of H2S on the oxygen consumption by the purified terminal oxidases bo3, bd-I, and bd-II from E. coli [92]. It turned out that the activity of cytochrome bo3 is quickly inhibited with an apparent half-maximal inhibitory concentration IC50 of 1.1 µM H2S (pH 7.4). This value is similar to the Ki of 0.55 µM H2S obtained by Nicholls and Kim for the beef heart cytochrome c oxidase at pH 7.48 [37]. The inhibition of the bo3 oxidase appeared to be fully reversible. The rapid and total recovery of the enzymatic activity occurs following the removal of H2S from the solution by the H2S scavenger, the Entamoeba histolytica O-acetylserine sulfhydrylase (EhOASS), in the presence of excess O-acetyl-L-serine (OAS). In contrast, neither the bd-I oxidase nor the bd-II oxidase is inhibited under identical conditions, even at a high concentration of H2S (58 µM) [92]. Similar results were obtained when examining the effect of H2S on the oxygen consumption by cell suspensions of the E. coli mutant strains which possess bo3, bd-I, and bd-II as the only terminal oxidase. The oxygen uptake by cells having bo3 as the sole oxidase is rapidly inhibited by 50 µM H2S. As in the case of the purified enzyme, the inhibition is promptly and completely restored after H2S depletion by the EhOASS/OAS system. Conversely, H2S at the same concentration does not affect the oxygen uptake by mutant cells having either bd-I or bd-II as the only oxidase [92]. Similar data on the E. coli membrane vesicles were reported by Korshunov et al. [93].
Forte et al. also tested if bd-I and/or bd-II oxidases, besides enabling aerobic respiration, promote the growth of E. coli cells in the presence of H2S [92]. A quantity of 200 µM H2S appeared to severely inhibit the growth of mutant cells with only cytochrome bo3. On the contrary, virtually no effect on cell growth was detected following the addition of H2S at the same concentration to the strains which express either bd-I or bd-II as the sole oxidase. Thus, in contrast with cytochrome bo3, which is potently and reversibly inhibited by H2S, both cytochrome bd-I and cytochrome bd-II are H2S-insensitive, and therefore, able to sustain respiration and cell growth in the presence of high levels of H2S. We assume that the mechanism of the inhibition of the E. coli cytochrome bo3 by H2S is very similar to that suggested for the H2S-mediated inhibition of another heme-copper terminal oxidase, mitochondrial complex IV ( Figure 5). Forte et al. also tested if bd-I and/or bd-II oxidases, besides enabling aerobic respiration, promote the growth of E. coli cells in the presence of H 2 S [92]. A quantity of 200 µM H 2 S appeared to severely inhibit the growth of mutant cells with only cytochrome bo 3 . On the contrary, virtually no effect on cell growth was detected following the addition of H 2 S at the same concentration to the strains which express either bd-I or bd-II as the sole oxidase. Thus, in contrast with cytochrome bo 3 , which is potently and reversibly inhibited by H 2 S, both cytochrome bd-I and cytochrome bd-II are H 2 S-insensitive, and therefore, able to sustain respiration and cell growth in the presence of high levels of H 2 S. We assume that the mechanism of the inhibition of the E. coli cytochrome bo 3 by H 2 S is very similar to that suggested for the H 2 S-mediated inhibition of another heme-copper terminal oxidase, mitochondrial complex IV ( Figure 5).
The H 2 S resistance of cytochrome bd is probably a key trait not only found in E. coli. Saini et al. [191] reported that H 2 S promotes the respiration and growth of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium bovis BCG. The authors concluded that the suppression of the cytochrome bcc-aa 3 supercomplex by H 2 S leads to the switching of the electron flow from MQ to cytochrome bd. The latter, in turn, stimulates respiration and ATP production, leading to the increased growth of the mycobacteria [191]. Furthermore, Kunota et al. [192] showed that multidrug-resistant and drug-susceptible clinical M. tuberculosis strains (but not non-pathogenic M. smegmatis) generate H 2 S endogenously, maintaining bioenergetic homeostasis by stimulating respiration primarily via the bd-type terminal oxidase. These findings are in agreement with the fact that the bd oxidases of the E. coli respiratory chain are H 2 S-insensitive [92,93].

Concluding Remarks
Accumulated evidence has shown that H 2 S is an effector molecule that controls energy metabolism. Depending on the concentration, H 2 S can either stimulate or inhibit the mammalian mitochondrial respiratory chain and F o F 1 -ATP synthase. An involvement of LDHA in the stimulatory effects of H 2 S on mitochondrial respiration has also been suggested. High H 2 S inhibits the bacterial respiratory chain terminating in cytochrome bo 3 but not cytochrome bd. The effects of H 2 S on a bacterial ATP synthase are still unknown. Although the role of H 2 S in microorganisms has been investigated less, there is currently an explosion of interest in the link between H 2 S and bacterial energy metabolism as a result of the recognition that this gaseous signaling molecule is critically important for the growth and colonization potential of pathogens, such as M. tuberculosis. A clearer picture of the effects of H 2 S on energy metabolism would represent a significant advance for the development of novel therapeutics.