Persulfide Biosynthesis Conserved Evolutionarily in All Organisms

Abstract

Significance: Persulfides/polysulfides are sulfur-catenated molecular species (i.e., R-S_n-R', n > 2; R-S_n-H, n > 1, with R = cysteine, glutathione, and proteins), such as cysteine persulfide (CysSSH). These species are abundantly formed as endogenous metabolites in mammalian and human cells and tissues. However, the persulfide synthesis mechanism has yet to be thoroughly discussed. Recent Advances: We used β-(4-hydroxyphenyl)ethyl iodoacetamide and mass spectrometry to develop sulfur metabolomics, a highly precise, quantitative analytical method for sulfur metabolites. Critical Issues: With this method, we detected appreciable amounts of different persulfide species in biological specimens from various organisms, from the domains Bacteria, Archaea, and Eukarya. By using our rigorously quantitative approach, we identified cysteinyl-tRNA synthetase (CARS) as a novel persulfide synthase, and we found that the CysSSH synthase activity of CARS is highly conserved from the domains Bacteria to Eukarya. Because persulfide synthesis is found not only with CARS but also with other sulfotransferase enzymes in many organisms, persulfides/polysulfides are expected to contribute as fundamental elements to substantially diverse biological phenomena. In fact, persulfide generation in higher organisms-that is, plants and animals-demonstrated various physiological functions that are mediated by redox signaling, such as regulation of energy metabolism, infection, inflammation, and cell death, including ferroptosis. Future Directions: Investigating CARS-dependent persulfide production may clarify various pathways of redox signaling in physiological and pathophysiological conditions and may thereby promote the development of preventive and therapeutic measures for oxidative stress as well as different inflammatory, metabolic, and neurodegenerative diseases. Antioxid. Redox Signal. 39, 983-999.

Keywords: CARS; CPERS; evolution; persulfide; polysulfide; supersulfide.

FIG. 1.

Evolutionary conservation of sulfur respiration in all organisms. Ancestral cells or organisms that initially appeared in the hydrothermal vent underwent sulfur respiration. Even ancient photosynthetic bacteria likely generated polysulfides such as S₈, which evolved and kept up with energy metabolic functions in the mitochondria of higher organisms during evolution. Transformation of the original respiration into a novel type of sulfur respiration occurred, which utilized sulfur-containing amino acid cysteine persulfides rather than inorganic S₈ as an electron acceptor. Our interpretation here is that an electron is transferred from sulfur to oxygen, so we call this process an S-O mixed-type hybrid respiration. This illustration presents the comprehensive history of sulfur respiration in the various organisms that live on earth. C. elegans, Caenorhabditis elegans.

FIG. 2.

H₂S in the hydrothermal vent: A fundamental component of energy metabolism of primitive organisms. One well-known major type of fundamental energy metabolism in organisms is mediated by sulfur, which is typically used by primitive organisms such as bacteria that live in the hydrothermal vent. These microorganisms, called chemolithotrophic bacteria, are also known as sulfur/sulfide-oxidizing bacteria—Thiobacillus. These bacteria can utilize H₂S as an electron donor to produce a proton gradient and energy via an ETC, which leads to sulfur allotropes such as S₈ and ATP production. ETC, electron transport chain; H₂S, hydrogen sulfide (Arnulf, 2006). Photographs taken from the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), https://www.jamstec.go.jp/e/ [accessed May 30, 2023].

FIG. 3.

Detection of protein persulfidation by using the PMSA. (A) Schematic drawing showing the reaction mechanism in the PMSA. In the initial step of the reaction, both thiol (-SH) and polysulfide (-S_n-H) groups of the cysteine residues in a protein are alkylated by electrophiles (E). In the second reaction step, the PEGylation probe BPM selectively attacks alkylated polysulfides at proximal sulfur atoms. Polysulfide-rich proteins acquire PEGylation and demonstrate low gel mobility. (B) Detection of protein persulfidation in the WT ADH5 by using the original PMSA. Numbers on the gels and blots indicate expected numbers of PEG moieties in the protein, which were inferred from the mobility shift distance. Proteins were detected by means of CBB staining. 8NcG, 8-nitro-cGMP; ADH5, alcohol dehydrogenase 5; ASBT, 2-aminosulfonyl benzothiazole; BPM, biotin-PEG-MAL; CBB, Coomassie Brilliant Blue; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTP, 4,4′-dithiopyridine; IAM, iodoacetamide; MBB, monobromobimane; MMTS, methyl methanethiosulfonate; MSBT, 2-methylsulfonyl benzothiazole, NEM, N-ethylmaleimide; PCMB, p-chloromercuribenzoic acid; PEG, polyethylene glycol; PMSA, PEG-conjugated maleimide labeled gel shift assay; WT, wild type.

FIG. 4.

Polysulfide hydrolysis promoted by electrophiles (E). Formation of an electrophile–sulfur adduct may initiate the heterolytic cleavage of the S-S bond mediated via the nucleophilic attack by water.

FIG. 5.

Sulfur metabolome and proteome analysis developed with HPE-IAM and LC-ESI-MS/MS. Schematic representation of the quantitative detection of LMW persulfides/polysulfides by using HPE-IAM as a trapping agent and LC-ESI-MS/MS analysis (upper panel) and quantitative detection of protein persulfides/polysulfides by means of trypsin digestion and LC-Q-TOF-MS (lower panel). ESI, electrospray ionization; HPE-IAM, β-(4-hydroxyphenyl)ethyl iodoacetamide; LC, liquid chromatography; LMW, low-molecular-weight; MS/MS, tandem mass spectrometry; Q-TOF, quadrupole time-of-flight.

FIG. 6.

Sulfur metabolic pathway coupled to translation by the CysSSH-producing enzyme CARS. CARSs produce CysSSH from cysteine. CARSs can convert cysteine to CysSSH via a PLP-dependent process using a second cysteine as the sulfur atom donor (independent of ATP and tRNA). The CARS-synthesized CysSSH can then form a tRNA-bound CysSSH adduct (also via CARS catalysis), which would result in the incorporation of CysSSH into proteins, thereby generating a protein containing a hydropersulfide function. The CysSSH-producing activity of CARSs is critically involved in translation-coupled protein persulfidation. CARS, cysteinyl-tRNA synthetase; CysSH, cysteine; CysSSH, cysteine persulfide; CysS-S_n-H, cysteine hydropersulfide/polysulfide; Cys-tRNA, cysteinyl-tRNA; PLP, pyridoxal phosphate.

FIG. 7.

Domain structure and key amino acid residues involved in the aminoacylation and PLP binding of CARS. General structure (upper panel) and conserved amino acid alignments (lower panel) of CARS from bacteria to humans. CARS1, cytosolic cysteinyl-tRNA synthetase; CARS2, mitochondrial cysteinyl-tRNA synthetase.

FIG. 8.

Scheme of Cys-tRNA^Cys formation in archaea. Sep, O-phosphoserine; SepCysS, Sep-tRNA:Cys-tRNA synthase; SepRS, phosphoseryl-tRNA synthetase.

FIG. 9.

Endogenous formation of persulfides in HEK293T cells. (A) Two different CARSs exist in mammals: CARS1 (cytosolic) and CARS2 (mitochondrial). (B) Intracellular levels of CysSSH in WT and CARS2 KO cells with CARS1 or CARS2 knocked down. Data are means ± SD (n = 3). **p < 0.01; N.S., not significant. (C) Production of CysSSH in CARS2 KO cells with WT or CARS2 C and K mutants added back. The data are means ± SD (n = 3). **p < 0.01 versus CARS2 KO mock. KO, knockout; SD, standard deviation; siRNA, small interfering RNA.

FIG. 10.

In vivo formation of sulfide species in WT and Cars2^+/− mice. Endogenous production of cysteine and CysSSH was identified by means of HPE-IAM labeling LC-MS/MS analysis in livers and lungs obtained from WT and Cars2^+/− littermates (21-week-old males). The data are means ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 11.

Endogenous protein polysulfidation in HEK293T cells. (A) The amounts of CysSSH formed in whole-cell protein recovered from WT and CARS2 KO HEK293T cells were quantified by using HPE-IAM labeling LC-MS/MS analysis. Data are means ± SD (n = 3). *p < 0.05. (B) Schematic drawing of the mechanism of the extramitochondrial release of CysSSH into the cytosol, which may regulate whole-cell protein polysulfidation.

FIG. 12.

Canonical and true pathways for persulfide biosynthesis. The canonical pathway for persulfide production consists of sulfotransferase enzymes (CBS and CSE; blue letters). CARS mediates the major, true pathway that governs persulfide biosynthesis (red letters). 3-MST and CBS/CSE may not be involved solely in sulfide production (black letters). 3-MST, 3-mercaptopyruvate sulfurtransferase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase.

FIG. 13.

Endogenous formation of reactive persulfide species and the function of these species in 8-nitro-cGMP metabolism. The reactive persulfide species-dependent metabolic pathway regulates 8-nitro-cGMP signaling. 8-Nitro-cGMP reacts with reactive persulfide species to form 8-SH-cGMP, with the release of nitrite. An additional reaction between the sulfhydrated metabolite 8-SH-cGMP and ROS forms cGMP by oxidative desulfidation, and this cGMP is then degraded by phosphodiesterase. cGMP, guanosine 3′,5′-cyclic monophosphate; 8-nitro-cGMP, 8-nitroguanosine 3′,5′-cyclic monophosphate; NO₂⁻, nitrite anion; ROS, reactive oxygen species.

FIG. 14.

Mechanisms to avoid oxidative damage to proteins caused by persulfides. A protein thiol (Protein-SH) is irreversibly oxidized by being excessively oxidized, but persulfides have a reducing ability and can provide a functionally reversible oxidation state, protein persulfidation (Protein-S_nH). The glutathione-thioredoxin system contributes to the reduction of excessively oxidized persulfides, perthiosulfinic acid (R-S_nSO₂H), and perthiosulfonic acid (R-S_nSO₃H).

FIG. 15.

Inhibition by CARS-generated persulfides/polysulfides of peroxidation and ferroptosis by scavenging radicals. RSSH, hydropersulfides.

FIG. 16.

Energy production in bacteria that utilizes sulfide. Chemolithotrophic sulfur-oxidizing bacteria such as Thiobacillus utilize H₂S as an electron donor to produce a proton gradient and produce energy via the ETC.

FIG. 17.

Mitochondrial sulfur respiration: Energy production by means of electron transfer conjugation of CARS2-derived persulfides. CysS-S_n-H produced by CARS2 in mitochondria may be reductively metabolized to sulfides and then oxidized by the SQR, in a manner linked to the ETC in mitochondria. CysS-S_n-H-dependent sulfur metabolism is coupled with formation of glutathione polysulfide (GS-S_n-H), which is controlled by the mitochondrial ETC. CARS2, mitochondrial cysteinyl-tRNA synthetase; ETHE1, ethylmalonic encephalopathy protein 1 (persulfide dioxygenase); GS-S_n-H, glutathione hydropersulfide/polysulfide; Q/QH₂, ubiquinone/ubiquinol; SQR, sulfide:quinone oxidoreductase; TCA, tricarboxylic acid; xCT, cystine/glutamic acid transporter.

FIG. 18.

Evolutionarily conserved persulfide biosynthesis in organisms. Persulfide synthesis regulates energy metabolism in the domain Bacteria, Archaea, and Eukarya. In eukaryotes, persulfides have various physiological functions, including anti-inflammatory effects, regulation of ferroptosis, and antiviral defense.