Hydrogen sulfide (H₂S), as an environmental toxin, is now confirmed to be a biological mediator and plays essential roles in normal physiology and in the responses to different stresses ^[1][2][1,2]. H₂S also regulates the responses to oxidative stress by interplaying with reactive oxygen species (ROS) at multiple levels ^[3][4][3,4] and protects mitochondrial function ^[5][6][5,6], maintaining mitochondrial homeostasis [7]. Mitochondria are the cells’ oxidation centers and power stations; they coordinate cell metabolism and immunity [8], and are both the source and the target of H₂S. H₂S can be produced inside or outside mitochondria, regulating mitochondrial energy metabolism, and maintaining mitochondrial functions under stress ^[3][9][3,9]. In mitochondria, the tricarboxylic acid (TCA) cycle is the final metabolic pathway of the three major nutrients (sugars, lipids, and amino acids) and the hub of the metabolism of sugars, lipids, and amino acids. The TCA cycle is a step in the process of respiration, after which high energy electrons are oxidized and ADPs are phosphorylated through the electron transport chain with the help of NADH, H⁺, and FADH₂ to produce ATPs [10]. The mitochondrial respiratory chain, also called an electron transfer chain, is a continuous reaction system composed of a series of hydrogen transfer reactions and electron transfer reactions arranged in a specific order; it produces the majority of ROS, and supplies the cell with energy [11]. Respiratory chain complex I (NADH-ubiquinone oxidoreductase) oxidizes NADH, pumps protons from the inside of the mitochondrial inner membrane to the membrane gap, and transfers electrons to ubiquinone; complex II (succinate dehydrogenase) has a role in transferring electrons from succinic acid to ubiquinone; complex III (ubiquinone-cytochrome c oxidoreductase), an essential mitochondrial protein complex in the oxidative phosphorylation process, transfers electrons from ubiquinone to cytochrome c; complex IV (cytochrome c oxidase) pumps protons into the membrane gap and transfers electrons from cytochrome c to oxygen. These protons drive ATP synthesis by ATP synthase [12]. Disorder in the mitochondrial respiratory complexes is an essential cause of mitochondrial disease and aging [13].

H₂S, as a colorless, corrosive gas, is poisonous and even lethal at high concentrations [14]; as a lipophilic molecule, it can diffuse readily through biological membranes. As a polar and hydrogen-bonding-capable molecule, H₂S has a membrane permeability comparable to O₂ and CO₂, which are nonpolar. H₂S can cross lipid bilayers with permeability coefficients from 0.5 cm/s to about 12 cm/s, which depend on the different membranes [15]. The solubility of H₂S in pure water is up to 3.846 mg/g at 20 °C, and aqueous H₂S is volatile due to its dissociation [16]. More than 80% of H₂S in the water at physiological pH is dissociated to hydrosulfide anion (HS⁻) and then dissociated to sulfide anion (S²⁻) at a higher pH, and the rest of the H₂S remains as an undissociated molecule [17]. H₂S, as a weak diprotic acid, has pKa₁ values of 6.98 at 25 °C and 6.76 at 37 °C [14]. Therefore, the availability of HS⁻ is high at neutral pH in vivo. The pKa values of the second dissociation are 17~19 at 25 °C [14]. Thus, alkaline sodium sulfide (Na₂S) and sodium hydrosulfide (NaHS) solution can be applied as H₂S sources to lower the pH.

The energies of orbitals for H₂S (−10.47 eV) are lower than those of HS⁻ (−2.31 eV), indicating that the nucleophilicity of HS⁻ is higher than that of H₂S [17]. With its negative charge and low electronegativity, HS⁻ can form a covalent bond with an electrophile (E⁺) by donating a pair of electrons, producing E-SH, and the product E-SH can also react with another E⁺ to form E-S-E [14]. This reactivity is the basis of the biological effect of H₂S.

The chemical properties of H₂S (or HS⁻) as nucleophiles give the possibilities for two kinds of interaction between H₂S and metals (Figure 1) [18]: (i) H₂S (HS⁻) can bind noncovalently or coordinate the transitional metals as a ligand; (ii) H₂S (HS⁻) can reduce the metal, accompanied by the production of HS^• and other downstream sulfur oxidation products. The positively charged transitional metal ions, such as iron and copper ions, can change valence by accepting an electron.

Cytochrome c oxidase (CcO), as a mitochondrial hemeprotein, contains copper centers, which are Cu_A and Cu_B, and ferric heme a and a₃ [19]. H₂S can bind to and reduce CcO and serve as an electron donor. Ferric heme a₃ can oxidize H₂S with low concentrations to be HS^•. The product HS^• is likely to react with HS⁻ to produce H₂S₂^•−. Alternatively, HS^• can be oxidized by oxygen to form HSOO^•. Despite inhibiting CcO, heme iron reduction promotes oxygen consumption, resulting in the stimulation of respiration ^[14][20][14,20]. At high levels, H₂S binds to the O₂-binding Cu_B center to be a Cu-SH complex that cannot be re-oxidized. Excessive H₂S coordinates to ferric heme a_3, forming a Fe-SH complex, eventually leading to irreversible inhibition of CcO. Cytochrome c has a similar behavior in that heme ferric iron is reduced by H₂S. Therefore, more reducing agents enter the electron transfer chain, consuming more oxygen. The inhibition of cytochrome c by H₂S, to some extent, promotes CcO reduction and respiration [21].

H₂S, a covalent hydride, is considered the simplest thiol, and its bond dissociation energy is about 385 kJ/mol, which is similar to that of the S-H bond in other thiols [22]. Both H₂S and HS⁻ act as reductants. H₂S can be oxidized by oxidants to substances with higher oxidation states, including sulfur (S⁰), sulfur dioxide (SO₂), sulfite (SO₃²⁻), sulfate (SO₄²⁻), sulfur trioxide (SO₃), and thiosulfate (S₂O₃⁻), and sulfonyl radical (HS^•).

H₂S can be oxidized by several biologically reactive species, such as nitrogen dioxide, hydroxyl radicals, peroxyl radicals, and superoxide radicals. The HS^• is the initial oxidation product of H₂S. HS^• can be transformed into SO₂^•− under the oxidation of O₂, while O₂ is catalyzed to be a superoxide radical (O₂^•−). O₂^•− can be dismutated by superoxide dismutase into O₂ and H₂O₂. The nucleophilic substitution of HS⁻ on H₂O₂ forms polysulfanes. Sulfenic acid (HSOH) formed by the reaction between HS⁻ and hydroperoxides (ROOH) can be transformed into HSSH by reacting with another HS⁻. The nucleophilic attack of HS⁻ on peroxynitrite (ONOOH) gives HSOH and NO₂⁻. ^•NO is involved in many vital physiological processes and signaling in mammals and plants and has complex crosstalk with H₂S signaling. H₂S can reduce ^•NO to form nitroxyl (HNO) or nitrososulfane (HSNO^•−), eventually leading to N₂O and sulfane sulfur formation [23]. Oxidization of ^•NO to NO₂⁻ can also be facilitated by H₂S. H₂S can stimulate the formation of S-nitrosothiols (RSNO) of cysteine caused by ^•NO.

In addition to S-nitrosothiols, persulfide (RSSH/RSS⁻) can be formed from the posttranslational modification of cysteines by H₂S (HS⁻). H₂S also reacts with GSSG to generate glutathione persulfide (GSSH) [24]. H₂S can transfer sulfur with the catalysis of sulfide quinone oxidoreductase (SQR) to GSH to form GSSH [25].

Apart from their similar characteristics to thiols, disulfides, polysulfides, and hydroperoxides, persulfides attract increasing attention in biology as versatile molecules. Compared with thiols and H₂S, persulfides are predicted to be more acidic and nucleophilic with a weaker S-H bond whose dissociation energy is 293 kJ/mol [26]. Thus, RSSH can reduce ferric cytochrome c to ferrous cytochrome c with the concomitant generation of RSS^•. The cysteine residues modify the sulfur transferase (ST) structures involved in the H₂S-producing process. The sulfur of the active site of the protein persulfides can be catalyzed by these enzymes to be thiols or sulfite. H₂S can react with protein sulfenic acids (RSOH) to form persulfides (RSSO₂H/RSSO₃H) [14]. Iron-sulfur (Fe-S) clusters, as inorganic cofactors, especially bind to respiratory complexes, becoming involved in fundamental life processes such as energy production as well as electron transfer. The generation of persulfides (RSSH) involves Fe-S cluster synthesis, which is the crucial step of Fe-S assembly in mitochondria [27]. Persulfides are unstable in solution at room temperature and react with the outer and inner sulfur, yielding sulfur and H₂S (HS⁻) [28]. Persulfides/polysulfides contain sulfane sulfur, which has six valence electrons and no charge [29] and is mainly responsible for the biological activity attributed to H₂S [30]. H₂S is synthesized by the same enzymes involved in forming sulfane sulfur [31], suggesting a close relationship between H₂S and sulfane sulfur and that these two reactive sulfur species always coexist ^[32][33][32,33]. It has been suggested that it is rather a sulfane sulfur, and not the H₂S itself, that acts as a signaling molecule and is responsible for the biological actions of RSS. The term H₂S is still used for narrative convenience in the following text.

Thus, H₂S can signal through reduction and/or direct binding of metalloprotein heme centers, potent antioxidants through reactive oxygen species/reactive nitrogen species scavenging, and modifying proteins through persulfidation [5].