The biosynthesis of H₂S includes both non-enzymatic and enzymatic pathways.

The non-enzymatic pathway of H₂S biosynthesis occurs due to the reaction of thiols or thiol-containing compounds with other molecules ^[1][2][3][4][18,29,30,31]. Glutathione (GSH) reduces inorganic polysulfides or hydrolyzed inorganic sulfide salts, i.e., sodium sulfide (Na₂S) or sodium hydrosulfide (NaHS) with water to produce H₂S ^[2][3][4][29,30,31]. Cysteine is the preferred substrate for the non-enzymatic production of H₂S, and the process is catalyzed by iron and vitamin B₆ ^[5][32].

The trans-sulfuration pathway is the primary source of endogenous H₂S and is the only way to produce endogenous cysteine, involving cellular sulfur metabolism and redox regulation ^[6][33] (Figure 1). Methionine, acting as a substrate, is catalyzed to produce cysteine eventually, with H₂S as the byproduct. Cystathionine-β-synthase (CBS, EC 4.2.1.22), cystathionine-γ-lyase (CSE, EC 4.4.1.1), cysteine aminotransferase (CAT, EC 2.6.1.3), and 3-mercaptopyruvate sulfurtransferase (3-MST, EC 2.8.1.2) are all involved in this pathway. CBS and CSE are only located in the cytoplasm, while CAT and 3-MST can be present in the cytoplasm and mitochondria ^[7][34]. In the first irreversible step of converting methionine to cysteine, CBS catalyzes the condensation of homocysteine with serine (Ser) or cysteine to form cystathionine. The main role of CSE in the trans-sulfuration pathway is in the conversion of cystathionine to cysteine and α-ketobutyrate. CBS can produce H₂S through β-substitution reactions ^[7][8][9][34,35,36]. Similarly, CSE can produce H₂S via the β-elimination reaction with cysteine or the γ-replacement reaction between two homocysteine molecules. There is also a mechanism for H₂S production, mediated by CAT and 3-MST in the transsulfuration pathway. CAT catalyzes l-cysteine and α-ketoglutarate to form 3-mercaptopyruvate (3-MP) and glutamate. The sulfur group of 3-MP is then transferred to 3-MST-accepting nucleophilic Cys247 in the presence of 3-MST, to produce 3-MST-bound persulfide and pyruvate. After this stage, the MST-persulfide reacts with thiols or is reduced by thioredoxin (Trx) to form H₂S ^[10][11][37,38]. In addition, 3-MST transfers the sulfur group from 3-MP to cyanide, to form thiocyanate ^[12][39]. Similar to the CAT/3-MST pathway, there is also a d-amino acid oxidase (DAO, EC 1.4.3.3)/3-MST pathway to generate H₂S (Figure 1). DAO metabolizes d-cysteine into 3-MP, which is metabolized into H₂S by 3-MST ^[13][40].

In plants, the synthesis pathway of H₂S can be divided into five types, according to the different substrates. These are cysteine degradation and sulfite reduction, cyanide detoxification, iron-sulfur cluster turnover, and carbonyl sulfide (COS) conversion ^[14][41] (Figure 1). Similar to animals, the metabolism of cysteine is the main source of endogenous H₂S production in plants. The plants also have a characteristic sulfate reduction assimilation of H₂S production. H₂S synthesis occurs in chloroplasts, cytoplasm, and mitochondria ^[15][42]. First, H₂S is mainly derived from cysteine degradation in the plant, catalyzed by different cysteine-degrading enzymes, including l-cysteine desulfhydrase (l-DES, EC 4.4.1.1), d-cysteine desulfhydrase (d-DES, EC 4.4.1.15), and l-3-cyanoalanine synthase (CAS, EC 4.4.1.9) ^[16][43]. Second, H₂S is derived from the reductive assimilation of sulfite (SO₃²⁻) in the plants (Figure 1). These two pathways of H₂S synthesis are closely linked. Sulfate or atmospheric sulfur dioxide (SO₂) is the source of SO₃²⁻ production in the plant in the presence of adenosine 5′-phosphosulfate (APS) reductase (APSR, EC 1.8.99.2). Atmospheric SO₂ can also produce SO₃²⁻ spontaneously via non-enzymatic interaction with water. Sulfite reductase (SiR, EC 1.8.7.1) reduces SO₃²⁻ to H₂S in the presence of chloroplast enzymes and ferredoxin ^[17][18][44,45]. Under alkaline conditions in the chloroplast stroma, plants spontaneously transport HS⁻ (a dissociated form of H₂S) into the cytoplasm (cytosol). With pyridoxal phosphate as a cofactor, l/d-cysteine is catalyzed by l/d-DES to produce pyruvate, NH₄⁺, and H₂S in the cytoplasm, chloroplasts, and mitochondria. Third, the nitrogenase Fe-S cluster-like (NFS/NifS-like) protein, which has similar activity to l-cysteine desulfurases, also catalyzes the conversion of cysteine to alanine and sulfur or sulfide using l-Cys as a substrate. It is also a possible source of H₂S in plants ^[19][20][46,47]. The cyanide detoxification mechanism is also an important source of H₂S in the plant (Figure 1). Ser and Acetyl-CoA are used to synthesize the intermediate reaction of O-acetyl-l-serine (OAS), catalyzed by Ser acetyltransferase (SAT, EC 2.2.1.30). O-acetyl-serine(thiol)lyase (OASTL, EC 2.5.1.47), also known as cysteine synthase, catalyzes the insertion of a particular sulfide (in this case, H₂S) into the carbon skeleton via an elimination reaction, and produces cysteine ^[21][48]. CAS involves cyanide detoxification and regulates the production of H₂S in mitochondria. H₂S is both an intermediate reduction product of sulfate assimilation and a substrate for the synthesis of cysteine. The biosynthesis of cysteine in plastids implies a transition between a reduction in the assimilated sulfate-reducing pathway and actual metabolism ^[21][48]. The iron-sulfur cluster located in Arabidopsis mitochondria is capable of assembling the NIF system and presents cysteine desulfurase activity, which may also offer a potential source of H₂S ^[22][49]. Besides the four H₂S synthesis pathways described above, carbonic anhydrase (CA, EC 4.2.1.1) catalyzes the hydrolysis of COS to produce carbon dioxide (CO₂) and H₂S. The plants absorb COS from the air and achieve the efficient use of sulfur assimilation via CA ^[23][24][50,51], which may also be a source of endogenous H₂S in the plant.

H₂S exhibits diverse physiological and signaling roles, mainly in four distinct biochemical ways (Figure 2): (1) reacting with reactive molecule species, such as ROS, reactive nitrogen species (RNS), hypochlorite (HOCl), and reactive carbonyl species (RCS) ^[25][26][27][52,53,54]; (2) binding to the metal center of metalloproteins or the reduction of the hemoglobin center ^[28][29][30][55,56,57]; (3) post-translationally modifying proteins with specific structures (e.g., proteins containing cysteine residues (-SH)), which are mainly via S-sulfhydration ^{[1][31][32][33]}[18,26,28,58]. The other PTMs are described in detail below ^[1][18]; (4) activities involving the oxidative and methylation pathways ^[6][34][33,59].

H₂S reacts with the reactive molecule species, including ROS and RNS ^[1][18] (Figure 2). H₂S can react with several biological oxidants, including superoxide radicals (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radicals (HO·), nitrogen dioxide (NO₂), peroxynitrite (ONOOH), and many others. H₂S reacts readily with HOCl to form polysulfides (-S-S_n-S-) ^[25][52]. Excessive ROS and RNS levels lead to oxidative stress when plants are exposed to adversity. In turn, H₂S significantly impacts the products of the plant’s defensive system. Nitric oxide (NO) is an important signaling molecule. H₂S can react with NO, leading to the formation of various nitrogen (nitrous oxide (N₂O), nitroxyl (HNO), S-nitrosothiols (RS-NO/SNO), and sulfur derivatives (e.g., S⁰, S⁻), which are thus involved in physiological signaling. NO converts the adversity-induced O₂^•− to the less toxic ONOO⁻. H₂S further reacts with ONOO⁻ to form thionitrate (HSNO₂) ^[27][54]. This property of H₂S to actively scavenge ONOO⁻ provides strong support for the inference that it synergizes with NO to reduce ROS oxidative stress. In addition, H₂S can react directly with NO to produce HNO and also react with RS-NO to form thionitrous acid (HSNO) ^[27][54]. HSNO can be metabolized to provide NO⁺, NO, and NO species, thus acting as a transportable NO reservoir in the organism that is involved in NO signaling.

H₂S binds to the metal centers of metalloproteins or participates in electron transfer ^[1][18] (Figure 2). H₂S involves the physiological regulation of the oxidative phosphorylation of the electron transfer chain (ETC) by means of binding to components of the ETC. It is mainly the direct binding of H₂S to COX that affects ETC function, and the reduction of COX by H₂S leads to the formation of HS^• or S^•− (which can interact with protein sulfhydryl groups (thiol)), affecting other components of the ETC. ETC complex IV, also known as COX, consists of two redox centers, Cyt a, Cu_A, and Cyt a₃, Cu_B. H₂S associates with the COX component, hematoxylin a₃ (heme a₃), and the Cu_B center, thus participating in the electron transfer of the ETC ^[28][29][55,56]. H₂S favors the formation of a polar environment (tyrosine (Tyr) residues and Cu_B centers) around the heme a₃ subunit, while H₂S promotes heme a₃ reduction to achieve an increase in COX enzyme activity at low concentrations. At high concentrations, H₂S can bind directly to the component a₃ and Cu_B centers of COX, resulting in the formation of the stable H₂S-Cu_B and unstable hemoglobin H₂S-Fe²⁺ inhibitory groups. In this case, the stability of the H₂S-Fe²⁺ group is dependent on H₂S concentration. However, the inhibition of COX by H₂S can behave differently, depending on the concentration. Unlike medium concentrations, a high concentration of H₂S is accompanied by the formation of stable hemoglobin a₃ H₂S-Fe³⁺ inhibitory groups, the inhibitory effect of which is irreversible ^[28][29][30][55,56,57]. In addition, the reduction of COX by H₂S promotes increased ATP synthesis (which can bypass complex III to promote ETC activity) and the accumulation of reactive sulfur ^[6][33].

H₂S can post-translationally modify proteins by converting the thiol group of cysteine residues to the persulfide group (-SSH) ^{[1][31][32][33]}[18,26,28,58] (Figure 2). This modification is named S-sulfhydration ^[1][18]. The increased nucleophilicity of the converted persulfides, compared to the thiol group, highlights the highly reactive nature of S-sulfhydration. This also explains the potential for persulfides to act as mediators of sulfide signaling.

The metabolic pathways of H₂S include oxidation and methylation ^[34][59] (Figure 2). The oxidation of H₂S occurs in the mitochondria and involves several enzymes, such as sulfide quinone reductase (SQR) and the ethylmalonic encephalopathy 1 protein (ETHE1, also known as persulfide dioxygenase), thiosulfate sulfurtransferase (TST, also known as rhodanese), and mitochondrial sulfite oxidase. SQR oxidizes H₂S in the inner mitochondrial membrane to produce persulfide species (e.g., glutathiol (GSSG)). At the same time, electrons released by SQR are captured by ubiquinone and transferred from H₂S to coenzyme Q and to ETC at complex III. The persulfide is further oxidized by ETHE1 to produce SO₃²⁻, which is further oxidized by sulfite oxidase to SO₄²⁻, or by TST to S₂O₃^{2− [34]}[59]. The metabolism of H₂S by methylation occurs more as a complementary mechanism to oxidation and takes place in the cytoplasm. Then, thiol S-methyltransferase converts H₂S to methanethiol (CH₄S) and dimethyl sulfide (CH₃)₂S, which is further oxidized by rhodanese to produce thiocynate and SO₄^{2− [6]}[33].