Protein persulfidation in plants: mechanisms and functions beyond a simple stress response

2.1 Protective effect of persulfides by enhanced oxidant scavenging

A crucial role of persulfides based on their chemical properties is to protect proteins from excessive and concomitant irreversible oxidation. The chemical properties of persulfides have been summarized in many excellent reviews (see e.g. Benchoam et al. 2019; Filipovic et al. 2018; Ono et al. 2014; Vignane and Filipovic 2023). Briefly, low molecular weight persulfides have a lower pK_a than the respective thiol resulting in predominant presence of the RSS⁻ anionic species at physiological pH (Benchoam et al. 2020; Cuevasanta et al. 2015). For example, a pK_a of 8.94 was determined for glutathione (GSH) whereas the pK_a of glutathione persulfide (GSSH) is 5.45 (Benchoam et al. 2020). In proteins, where the molecular microenvironment impacts on the reactivity of redox-sensitive cysteine residues, this correlation may not always hold true (Trost et al. 2017). The persulfidated form of human thiosulfate sulfurtransferase, for instance, has a pK_a of 9.38, whereas that of the thiolated form is 6.5 (Benchoam et al. 2024). Furthermore, persulfides are more nucleophilic than the respective thiols. This persulfide nucleophilicity is based on the presence of a sulfur atom with free electron pairs that increases the nucleophilicity of the outer or terminal sulfur atom via the so-called α-effect (Benchoam et al. 2020; Ida et al. 2014). These two characteristics, lower pK_a and higher nucleophilicity, result in a faster reactivity with two-electron oxidizing electrophiles such as H₂O₂ and peroxynitrite or one-electron oxidants like peroxyl radicals compared to the respective thiol (Chauvin et al. 2016; Cuevasanta et al. 2015; Ida et al. 2014). Hence, due to the enhanced oxidant scavenging of persulfides, especially low molecular weight molecules like GSSH, they can be considered to have a protective effect. GSSH for example, reacts 22 times faster in vitro with oxidizing H₂O₂ and even 97 times faster with peroxynitrite compared to GSH (Benchoam et al. 2020). In addition, it was shown in vitro that GSSH reduces radicals. Forming stable perthiyl radicals (GSS•) and recombining into tetrasulfides (GSSSSG), GSSH prevents the propagation of radical chain reactions, thereby eliminating radicals from the system. This was further confirmed in vivo where increased amounts of persulfides result in decreased radical amounts and radical-driven lipid peroxidation (Barayeu et al. 2023b).

It should be noted though that the described faster reactivity of persulfides with oxidants compared to the respective free thiol cannot be generalized. Exceptions of antioxidant enzymes exist where persulfidation of the active site cysteine results in a decreased reduction of specific endogenous substrates. This was shown for MtAhpE, which in its persulfidated form was found to be less reactive towards H₂O₂ and peroxynitrite (Cuevasanta et al. 2019; Reyes et al. 2018). Peroxidatic cysteines of peroxidases are characterized by a low pKa, which is achieved through stabilization of the thiolate form by surrounding amino acids that depends on the correct topology of the active site. Based on molecular dynamic simulations, it was suggested that persulfidation in this case results in significant disruption in the topology of the active site altering key interactions involved in catalysis of the Prx substrates (Cuevasanta et al. 2019).

2.2 Protective effect of persulfides for protein thiols

In vivo, several studies show a direct connection between elevated H₂O₂ and protein persulfidation levels. In Arabidopsis, it was shown that under non-photorespiratory conditions both, H₂O₂ levels as well as protein persulfidation levels, increase (García-Calderón et al. 2023). Also, in mouse embryonic fibroblasts and HeLa cells an increased amount of persulfides was detected after H₂O₂ treatment (Zivanovic et al. 2019). Notably, H₂S cannot directly react with a cysteine thiol but needs a preceding oxidation step such as sulfenylation. The reaction of sulfide with sulfenic acids is considered to render H₂S as a cellular defense against cysteine overoxidation (Figure 1A). While the formation of sulfenic acid is a reversible key reaction in cellular redox signaling, the subsequent oxidation to sulfinic (Cys-SO₂H) or sulfonic (Cys-SO₃H) acid would render the oxidation irreversible and thus result in permanent inhibition of the respective protein, although some exceptions exist for reduction of sulfinylated proteins, particularly through ATP-dependent reduction by sulfiredoxins (Biteau et al. 2003; Iglesias-Baena et al. 2011; Woo et al. 2003). In contrast, enzymatic activity of persulfidated proteins can be recovered after oxidation. The respective persulfide derivatives perthiosulfenic acid (Cys-SSOH), perthiosulfinic acid (Cys-SSO₂H), and perthiosulfonic acid (Cys-SSO₃H) can be reduced back by the thioredoxin (TRX) system or other reductants that attack the disulfide bridge as it was shown in vitro for HSA and peroxiredoxin Prx2 as well as for the 3-mercaptopyruvate sulfurtransferases (MSTs) from rat and Arabidopsis after H₂O₂ treatment (Dóka et al. 2020; Moseler et al. 2021; Nagahara et al. 2012). Also, in bovine and human cells, in which the TRX reductase was downregulated or inhibited chemically by auranofin, an increased amount of persulfidated proteins is detected (Dóka et al. 2016; Wedmann et al. 2016). In human cells, a large number of proteins are perthiosulfenylated even though they are not facing elevated H₂O₂ levels resulting from activation of NADPH oxidases DUOX1 and NOX2. This suggests possible functions of persulfide oxidation beyond a protective role of persulfidation (Heppner et al. 2018). The role of this persulfide derivative in redox signaling, however, awaits further characterization.

Figure 1:

Impact of persulfides on proteins. (A) Protection against overoxidation. Under oxidative stress, cysteine thiols can be oxidized to sulfenic acid. This reaction is reversible, while the further oxidation to sulfonic acid is irreversible. Thiols can get persulfidated either enzymatically (indicated with P) or sulfide (HS⁻) reacts with sulfenic acid. Because of the intrinsic disulfide bridge the oxidized persulfides can get reduced back by enzymes such as thioredoxins (indicated by scissors) resulting in recovery of the native thiol. (B + C) Regulation of enzymatic activity. The persulfidation can occur outside of the active site causing conformational changes of the protein (B). These changes can result in different outcomes like enhancing protein-protein interaction, substrate affinity, subcellular localization or oligomerization. On the other hand, persulfidation can occur directly at the active site cysteine (C), where it can have inhibitory or enhancing effects.

Besides the reduction of persulfide derivatives and subsequent recovery of the free thiol moieties, several examples for proteins exist, whose persulfidated form provides protection against oxidation. For the cytosolic pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase 6 (G6PD6) it was shown that persulfidation of a conserved cysteine (C159 in Arabidopsis and C155 in tomato) alleviates oxidative protein inactivation caused by elevated levels of H₂O₂ under salt stress. On the one hand, the persulfidation induces structural changes in the G6PD6 homotetramer that support stabilization of enzyme activity and promote the affinity to NADP⁺. On the other hand, the persulfidation mitigates oxidative modifications of G6PD6 at C159/C155 that would result in enzyme inhibition (Wang et al. 2023).

In addition, persulfide levels are linked to protective physiological effects. For the antioxidant N-acetylcysteine, it was shown that its cytoprotective effect is based on the generation of supersulfides predominantly in mitochondria (Ezeriņa et al. 2018; Pedre et al. 2021). Furthermore, it was shown in human fibroblasts, Caenorhabditis elegans and mice that organisms with elevated persulfide levels had a longer lifespan compared to the respective control (Zivanovic et al. 2019). Additionally, in older individuals decreased amounts of persulfides but increased sulfinylation levels were detected supporting the idea that increased persulfidation improves the capacity to cope with stress stimuli and reactive oxygen species-related aging (Redman et al. 2018; Zivanovic et al. 2019). Arabidopsis null mutants lacking the L-cysteine desulfhydrase DES1 are characterized by a decreased sulfide content and a decreased level of protein persulfidation (Álvarez et al. 2010, 2012a; Aroca et al. 2017a). Phenotypically, these biochemical parameters correlate with premature leaf senescence. While so far only correlative, these observations may pave the way towards molecular analysis of the underlying mechanistic processes.

2.3 Identification of persulfidated cysteines and impact of persulfidation on protein activity

Basal protein persulfidation comprises nearly 5 % of the Arabidopsis proteome under non-stress conditions indicating other roles of persulfidation beyond mere protection of cysteines against oxidation (Aroca et al. 2017a; García-Calderón et al. 2023; Jurado-Flores et al. 2023). Several methods are available to detect persulfides in vivo based on their nucleophilic nature; each of them facing limitations like specificity or sensitivity (reviewed in Filipovic et al. 2018; Huang and Xie 2023; Pantaleno and Scuffi 2024). To detect protein persulfides, thiols and persulfides are frequently first alkylated by biotin-labeled alkylating agents like EZ-Link Iodoacetyl-PEG2-Biotin and pulled down from the protein mixture. Subsequently persulfides are separated in a reduction step leaving the non-reducible thiol-tagged biotin conjugates bound to the streptavidin beads (Dóka et al. 2016, 2021; Longen et al. 2016). Some alkylating agents like N-ethylmaleimide and iodoacetamide are, however, unsuitable as they convert the persulfides into thioethers (Schilling et al. 2022, 2023). The currently preferred method to detect protein persulfidation is the dimedone-switch method (for a detailed protocol see Aroca et al. 2022 and Zivanovic et al. 2019). The disadvantage of both methods is that they do not identify the persulfidated cysteine itself, but only the protein, which leaves ambiguities regarding the persulfidated thiols if more than one cysteine is present.

To identify specific persulfidated cysteine residues and their effect on enzyme activity, often recombinant proteins or cell lysates are treated with sulfide salts like NaHS and Na₂S or other sulfide donors like morpholino–phosphino–dithioic acid (GYY4137). These sulfide donors initially form polysulfides in solution, which subsequently are most likely the source for persulfidation of the respective cysteines rather than the sulfide donor itself (Greiner et al. 2013). Whether the thiol modification is specific for distinct cysteine thiols or also occurs non-specifically on other thiols needs to be demonstrated in vivo. A prominent example for a protein that is prone to artificial persulfidation is the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For GAPDH from mouse liver lysates or recombinant human GAPDH treated with NaHS, it was shown that persulfidation of the catalytic cysteine C150 enhances the GAPDH activity (Mustafa et al. 2009). Similar treatment of recombinant human GAPDH with NaHS resulted in persulfidation of C247, whereas treatment with potassium polysulfide resulted in persulfidation of C247 and C156. The cysteine residue C152, which corresponds to C150 in mouse GAPDH, was not persulfidated in this case. After treatment with polysulfide, human GAPDH did not show an increased but rather a decreased activity (Jarosz et al. 2015). Hence, apart from improving specificity and sensitivity, another prerequisite for the detection of biologically meaningful protein persulfidation is the application of methods that directly identify persulfidated cysteine residues. This prerequisite is fulfilled by the quantitative thiol reactivity profiling (low pH-QTRP) method in which the reduction step after alkylation is omitted to retain the labeled probes during digestion into tryptic peptides. Probe-labelled peptides are subsequently conjugated with a biotin-tag, captured by streptavidin beads and analyzed by mass spectrometry (Fu et al. 2020).

The effect of persulfidation on enzyme activity depends on the protein and the complexity of persulfide-induced changes bars making general statements about the respective consequences. The persulfidation can occur outside the active site and induce conformational changes (Figure 1B). These changes can attenuate the binding of the respective proteins to interaction partners, regulate oligomerization, affect substrate affinity or enhance certain subcellular sequestration (Aroca et al. 2017b; Chen et al. 2020; Ma et al. 2023; Wang et al. 2023). Similar to C156 in the human GAPDH, also the homologous residue C160 in the Arabidopsis orthologue GAPC1 can get persulfidated. NaHS treatment of Arabidopsis plants resulted in the persulfidation of the protein and translocation to the nucleus (Aroca et al. 2017b). A similar nuclear sequestration was also observed in cadmium-stressed plants suggesting a role of nuclear GAPC1 in oxidative stress signaling or protection (Vescovi et al. 2013). OPEN STOMATA 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6) is expressed in guard cells and involved signaling pathways mediating stomatal closure. Two cysteines (C131 and C137) adjacent to the catalytic loop of the kinase were identified to be persulfidated in vitro after incubation of recombinant SnRK2.6 with either NaHS or the alternative sulfide donor GYY4137 (Chen et al. 2020). Furthermore, the persulfidation of SnRK2.6 was confirmed in Arabidopsis ost1 null mutants by analyzing the differential SnRK2.6 persulfidation levels after complementation of ost1 with wild-type SnRK2.6 or a mutated SnRK2.6^{C131S C137S} variant. Persulfidation alters the SnRK2.6 structure bringing the pivotal phosphorylation site S175 closer to the phosphate-acceptor D140, which increases the kinase activity (Chen et al. 2020, 2021). Cys/Ser substitution variants expressed in the mutant background were not able to complement the drought-sensitive phenotype of ost1 indicating an essential role of both Cys residues in regulating the protein activity (Chen et al. 2020, 2021). An example for persulfidation affecting the binding to interaction partners was shown recently for the transcription factor WRKY6 in tomato (Zhang et al. 2024). Persulfidation of WRKY6 at C396 attenuates binding to the W-box in the promoter of the chlorophyll degradation-related gene SGR1 and the senescence-related gene SAG12 in unripe tomato. During fruit ripening H₂S levels decrease and concomitantly the degree of WRKY6 persulfidation also decreases. Subsequently, the enhanced binding to the promotor of the respective WRKY6 target genes results in increased SGR1 and SAG12 expression, which leads to fruit ripening and plant senescence. Expression of WRKY6 C396A in the wrky6 background resulted in enhanced expression of SGR1 and SAG12 accompanied with early fruit ripening. On the other hand, increasing H₂S levels either exogenously by fumigation or endogenously by overexpression of the cytosolic H₂S-generating enzyme L-cysteine desulfhydrase (LCD1) significantly delayed tomato fruit ripening indicating that persulfidation is important for the regulation of fruit ripening (Zhang et al. 2024).

Beside persulfidation of proteins outside their catalytic center, active site cysteines can get persulfidated resulting in increased or decreased activity (Figure 1C). The autophagy-related (ATG) Cys protease AtATG4a is persulfidated at C170 that is part of the catalytic Cys-His-Asp triad. This persulfidation inhibits AtATG4 activity based on conformational changes and intramolecular rearrangements of the catalytic site (Laureano-Marín et al. 2020). Also, for S. cerevisiae and Chlamydomonas reinhardtii ATG4 orthologues a redox regulation was shown, but so far no connection to persulfidation was made (Pérez-Pérez et al. 2014, 2016). For the cytosolic ascorbate peroxidase 1 (APX1) persulfidation at C32 was detected, which results in an increased activity (Aroca et al. 2015). Treatment of tomato plants with NaHS also increased APX activity in the respective crude extracts (Li et al. 2020).

2.4 Persulfidated proteins in Arabidopsis mitochondria

Although little is known about the effects of persulfidation on mitochondrial proteins, more than 80 mitochondrial proteins were identified in the reported Arabidopsis persulfidome (García-Calderón et al. 2023; Jurado-Flores et al. 2023) (Table 1). Mitochondria are the hub for central metabolic processes including the tricarboxylic acid cycle (TCA cycle), amino acids biosynthesis, as well as the production of several cofactors like iron sulfur cluster, lipoic acid or the precursor of molybdenum cofactor. Hence, a careful and precise regulation of metabolic dynamics in order to adapt metabolism and signal transduction has to be considered. The current proteomic data for the persulfidome in plants represents a valuable starting point for deciphering the actual targets of persulfidation and to further elucidate the molecular function of this cysteine modification.

Based on mitochondrial functions, it is not surprising that a global KEGG analysis (at least 7 proteins have to cluster) of the respective proteins associate them with glyoxylate metabolism, pyruvate metabolism, the TCA cycle, oxidative phosphorylation or the propanoate pathway (Figure 2A). A more detailed gene ontology (GO) analysis of the biological processes (at least 3 proteins have to cluster) indicate that nearly 50 % of the proteins are involved in amino acid catabolism, pyruvate metabolism or cellular respiration (Figure 2B). The other GO categorizations revealed that the persulfidated proteins play a role in several important biological processes like photorespiration. Also, the GO term ‘response to cadmium (Cd) ion’ is present.

Figure 2:

Functional classification of persulfidated proteins localized to mitochondria. (A) KEGG pathways associated to the mitochondrial proteins listed in Table 1. At least seven proteins had to cluster to represent a pathway. (B) Functional classification of gene ontology terms categorized by biological processes. At least three proteins had to cluster to define a group. All results were created with ClueGO v2.5.10 using Cytoscape (https://cytoscape.org/).

This can potentially be linked to previous reports in which Arabidopsis mutants lacking mitochondrial D-cysteine desulfhydrase (DCDES1, see 3.4 D-cysteine desulfhydrase), which is involved in H₂S production, where shown to be more sensitive to Cd, while transgenic lines overexpressing DCDES1 were more tolerant to Cd stress when compared with wild-type plants (Zhang et al. 2020). Under normal conditions, the content of H₂S was similar in wild-type plants and dcdes1 null mutants, but H₂S production was impaired in dcdes1 plants under cadmium stress. Cd stress stimulated the expression of DCDES1 in a process mediated by the Cd-induced transcription factor WRKY13, which binds to the DCDES1 promoter (Zhang et al. 2020). Also in alfalfa (Medicago sativa) increased DCDES activity was observed in less than 1 h after exposure to Cd-stress (Cui et al. 2014).

3.1 β-Cyanoalanine synthase

A pathway for H₂S production present in plants and bacteria is the conversion of cyanide and cysteine into β-cyanoalanine and H₂S catalyzed by the pyridoxal 5′-phosphate (PLP)-dependent β-cyanoalanine synthase CAS-C1 (syn. CYSC1; At3g61440; EC 4.4.1.9) (Arenas-Alfonseca et al. 2018; Machingura et al. 2016; Yamaguchi et al. 2000). Also, in some arthropod lineages, like Lepidoptera and some mites, a gene with putative CAS function was identified (Wybouw et al. 2014). Closely related to O-acetylserine (thiol) lyases, that synthesize cysteine from O-acetylserine and sulfide, CAS enzymes from various plant species (Arabidopsis, spinach, lupins, potato, rice) unanimously favor CAS activity over cysteine synthase activity (Hatzfeld et al. 2000; Hendrickson and Conn 1969; Lai et al. 2009). Based on a crystal structure of soybean CAS, this functional specialization was linked to structural differences in the PLP binding sites of these enzyme classes (Yi et al. 2012). Mitochondria are the major target of cyanide which inhibits cytochrome c oxidase (COX) by irreversibly binding to the heme iron (Antonini et al. 1971). In plants, cyanide derives from either the catabolism of cyanogenic glycosides or from ethylene biosynthesis, where cyanide is a degradation product of 1-aminocyclopropane-1-carboxylate (ACC) (Siegień and Bogatek 2006). Interestingly, it was shown in tomato that persulfidation of ACC oxidases 1 and 2 negatively regulates ethylene biosynthesis (Jia et al. 2018). Plants harness the toxicity of cyanide for protection against herbivores by hydrolyzing cyanogenic glycosides upon herbivory damage (Machingura et al. 2016). In Brassicaceae, the degradation of glucosinolates can also contribute to cyanide generation (Machingura et al. 2016). The main pathway for cyanide detoxification occurs via the mitochondrial CAS-C1 (Machingura et al. 2016). The cas-c1 null mutant has no obvious shoot phenotype but is defective in root hair formation and accumulates cyanide in root tissues. The observed root hair phenotype is likely due to the accumulation of cyanide since the root hair defect is phenocopied in WT plants treated with cyanide. Furthermore, an increase of the alternative oxidase pathway in the cys-c1 mutant was observed, indicating an inhibition of COX by cyanide (García et al. 2010). Considering the high abundance of the protein in mitochondria, but low cyanide concentrations, since the IC₅₀ for COX is 7 nM cyanide, CAS-C1 is probably a key enzyme for cyanide detoxification rather than supersulfide generation (Birke et al. 2012; Fuchs et al. 2020). No information, however, is available to which extent this pathway contributes to supersulfide generation leaving this question open for further investigations.

3.2 3-Mercaptopyruvate sulfurtransferases

3-Mercaptopyruvate sulfurtransferases (EC 2.8.1.2, MSTs) belong to the sulfurtransferase (STR) family and contain two characteristic rhodanese domains connected by a short linker peptide (Spallarossa et al. 2004). The active site locates at the interface between these domains and the catalytic cysteine residue is part of a canonical CG[S/T]GVT motif within the C-terminal domain (Yadav et al. 2013). Across kingdoms, MSTs are highly conserved and in Arabidopsis represented by two sulfurtransferases MST1 (syn. STR1; At1g79230) in mitochondria and MST2 (syn. STR2; At1g16460) in the cytosol (Nakamura et al. 2000; Moseler et al. 2020). All photosynthetic organisms (including cyanobacteria) possess at least one MST. In all cases where only one gene coding for MST is present, the MST contains an N-terminal extension that is predicted to function as a target peptide for mitochondrial localization (Moseler et al. 2020). The cytosol and the mitochondria of human cells both contain an MST (syn. tRNA thiouridin modification protein 1, TUM1) which are derived from two splice variants of the same gene (Fräsdorf et al. 2014). While good evidence exists for the function of MSTs in supersulfide generation in bacteria and vertebrates (Lec et al. 2018; Pedre and Dick 2021; Pedre et al. 2023; Yadav et al. 2020), little is known about their counterparts in plants. Arabidopsis mst1 null mutants show an ambiguous shrunken seed phenotype with ∼87 % of embryos arrested at heart stage (Mao et al. 2011). The remaining 13 % mature without any phenotypic aberrations compared to wild-type controls. In contrast, mst2 null mutants generally appear like the wild type (Mao et al. 2011; Nakamura et al. 2000). The mst1 mst2 double mutant, however, is embryonically lethal at heart stage indicating functional redundancy of both MSTs in an essential metabolic process (Mao et al. 2011).

In vitro, Arabidopsis MSTs catalyze the desulfuration of 3-mercaptopyruvate (3-MP) to pyruvate while generating a persulfide on their catalytic cysteine (Moseler et al. 2021). Subsequently, the outer sulfur atom of the persulfide is transferred to an acceptor molecule, either a low-molecular weight thiol or a thiol-containing protein. So far, protein-protein interactions of Arabidopsis MSTs were observed only with TRXs through bimolecular fluorescence complementation (BiFC) (Henne et al. 2015). The presumed transsulfuration reaction from the MST persulfide to the dithiol TRX and the subsequent substitution of sulfide by the second TRX cysteine should lead to the formation of H₂S. This principal possibility has been demonstrated with the release of H₂S from 3-MP in the presence of the TRX system comprising the TRX, NADPH-dependent thioredoxin reductase (NTR) and NADPH or other reductants in vitro (Figure 4) (Moseler et al. 2021). Besides TRXs, low molecular weight thiols like glutathione or cysteine can also act as sulfur acceptors of MSTs (Höfler et al. 2016; Kimura et al. 2017; Moseler et al. 2021). For Arabidopsis MST1, the catalytic efficiency with mitochondrial TRXo1 as acceptor was, however, 100-fold higher when compared to GSH (1.3 × 10⁶ M⁻¹ s⁻¹ and 2.7 × 10⁴ M⁻¹ s⁻¹, respectively) (Moseler et al. 2021). The binding affinity between MST1 and TRXo1 (K _m  = 5.3 µM) falls into a physiological meaningful range and compares well with that of human MSTs (MPST1 and MPST2) with thioredoxin TXN (Yadav et al. 2020). GSH concentrations in mitochondria of plant cells are in the low millimolar range (Fricker et al. 2000) and exceed the binding affinity between MST1 and GSH (K _m  = 200 µM), suggesting that MST1 is continuously saturated with GSH to generate GSSH. However, the total pool of accessible protein thiols in mitochondria may reach concentrations comparable to that of GSH (Requejo et al. 2010). Considering the higher binding affinities of MSTs to protein clients (Moseler et al. 2021; Yadav et al. 2020), an MST-mediated protein persulfidation is thus possible despite comparatively high GSH concentrations. Indeed, for MST from S. cerevisiae, it was shown that protein clients are the most efficient sulfur acceptors in vitro, with the exception of sulfite (Pedre et al. 2023). In the absence of other putative interaction partners, both Arabidopsis MSTs can transfer the persulfide to redox-sensitive green fluorescent protein 2 (roGFP2) revealing them as putative protein persulfidases (Moseler et al. 2021). Recently, it was shown that partial depletion of MST in human cells significantly lowers cellular protein persulfidation levels (Pedre et al. 2023). Seven of the 64 identified persulfidation targets of human MST are predicted to be localized in mitochondria (Consortium et al. 2023; Pedre et al. 2023). All seven human proteins have homologs in Arabidopsis with five of them sharing conserved cysteines. One of these, isocitrate dehydrogenase (IDH3A) was also found to be persulfidated in Arabidopsis, highlighting potential similarities in persulfidation pattern (García-Calderón et al. 2023; Jurado-Flores et al. 2023). Also in the heart tissue of MST knock-out mice, a reduced persulfidation of 24 proteins was found (Drekolia et al. 2024). Comparison with the mitochondrial Arabidopsis persulfidome (Table 1) revealed several homologs to be persulfidated also in Arabidopsis like the malate dehydrogenases (MDH) or succinate dehydrogenase (SDH1-1).

Figure 4:

The putative role of Arabidopsis MST1 in persulfide formation. Cysteine is deaminated to 3-mercaptopyruvate (3-MP) by a yet unknown cysteine transaminase (CTA). Subsequently, MST1 subtracts the sulfur from 3-MP by forming a persulfide on its active site cysteine along with the release of pyruvate. The persulfide can then be transferred to several acceptors. One possibility are low molecular weight molecules like GSH to form glutathione persulfide (GSSH). Other presumed persulfide acceptors are proteins (P), but so far, no evidence for direct persulfidation of distinct proteins exist except for thioredoxins (TRX). In the latter case, the persulfide is transferred from MST1-SSH to the N-terminal cysteine in the CxxC active site cysteine of the TRX. Subsequently, the C-terminal resolving cysteine of the active site motif attacks the persulfide to release H₂S and oxidized TRX. The TRX disulfide is then reduced by thioredoxin reductases (NTRA/B) with NADPH + H⁺ as their electron source. H₂S can react with sulfenylated (-SOH) proteins resulting in the protein persulfidation. Excess H₂S can be detoxified via OAS-TL c, which converts sulfide and O-acetylserine to cysteine. Green indicates the sulfur mobilization, blue the persulfide transfer and red the sulfide fixation.

Plant MSTs thus are candidates to produce H₂S and also candidates to act as persulfidases that persulfidate proteins or LMW molecules without releasing H₂S. The presence of two isoforms, MST1 and MST2, in Arabidopsis in two compartments and yet partially redundant function calls for further genetic and biochemical characterization of these enzymes to clarify their function in vivo.

3.3 Cysteinyl-tRNA synthetases

Other highly conserved enzymes related to supersulfide formation in both cytosol and mitochondria are the cysteinyl-tRNA synthetases (CARSs, Cysteine-tRNA ligase; EC 6.1.1.16) (Ogata et al. 2023). Generally, CARS plays a role in protein synthesis by producing cysteinyl-tRNA using cysteine and aminoacyl-tRNA as substrate. CARS can, however, also convert cysteine to Cys-SSH and Cys-S(S) _n H using cysteine as sulfur donor, which is described as cysteine persulfide synthase (CPERS) activity of CARS (Akaike et al. 2017). In Arabidopsis, three CARSs are present. CARS1 (At3g56300) and CARS2 (At5g38830) are cytosolic, CARS3 (syn. SYCO; At2g31170) is dual-localized in plastids and mitochondria, and, in addition, a splice variant is predicted to code for a cytosolic isoform (Peeters et al. 2000). While there is no information on the role of CARS1 and CARS2, an essential function of CARS3 in plant development is evident from embryo lethality of homozygous cars3 null mutants. This appears to be consistent with the situation in mice where deletion of mitochondrial CARS2 is lethal (Akaike et al. 2017). In Arabidopsis, the lethality is based on the inability of the two haploid polar nuclei to fuse in the central cell of the female gametophyte (Kägi et al. 2010). Furthermore, cars3 mitochondria of the central cell lacked regular cristae underpinning the role of mitochondria during nuclear fusion (Kägi et al. 2010; Portereiko et al. 2006).

Bacterial CARS as well as mitochondrial CARSs from human and mice are able to generate cysteine persulfides using a PLP-cofactor (Akaike et al. 2017). In this reaction, the PLP-cofactor is coordinated by lysine residues including a ₁₂₄KIIK₁₂₇ and a ₃₁₇KMSK₃₂₀ motif (numbering is based on human CARS2). In Arabidopsis CARS3, the KMSK motif is conserved while the KIIK motif is converted to KIIA. Although only the residues K124 and K317 are involved in PLP-binding, it was shown for Escherichia coli CARS that individual K-to-A substitutions of all four lysine residues have no effect on protein synthesis but result in decreased CPERS activity (Akaike et al. 2017). This observation leads to the question whether the corresponding Arabidopsis protein does have CPERS activity at all? Nevertheless, in yeast or zebrafish CARS2, the PLP-binding site reads KIIL or KIIQ rather than the KIIK (Ogata et al. 2023). Yeast CARS2 was shown to exhibit a CPERS activity indicating some possible variations in the motif (Nishimura et al. 2024).

In S. cerevisiae, mutation of the first lysine in the KIIK motif to alanine causes the same enzymatic changes observed in E. coli. Notably, this yeast mutant showed an impaired mitochondrial energy metabolism with decreased ATP levels (Nishimura et al. 2024). Also, for human cells, the knockdown of mitochondrial CARS2, which resulted in decreased Cys-SSH content, alters mitochondrial morphogenesis and bioenergetics. Knockdown of cytosolic CARS1, however, showed no significant decrease of Cys-SSH. The observations together demonstrate that the formation of Cys-SSH occurs more frequently in mitochondria (Akaike et al. 2017). An interesting aspect is that Cys-SSH generated by CARS2 in human cells can serve as direct acceptor for electrons from the mitochondrial electron transport chain (ETC) resulting in HS⁻ production. Induction of mitochondrial DNA deprivation as well as specific inhibition of complex III by antimycin A decreased HS⁻ levels, indicating that Cys-SSH is directly reduced by electrons from the ETC. HS⁻ is subsequently used by sulfide quinone oxidoreductase (SQR) generating GSSH, which can be further oxidized to sulfite by the persulfide dioxygenase ETHE1 (ETHYLMALONIC ENCEPHALOPATHY 1). On the one hand, this mechanism prevents excessive generation of ROS by leaked electrons from the ETC and on the other hand it prevents sulfide toxicity (Akaike et al. 2017; Barayeu et al. 2023a). In plants, however, SQR is not present excluding thereby this supersulfide-mediated electron flow (see Section 4. Protein depersulfidation and refixation of sulfide).

A new aspect that was introduced by Akaike and colleagues is that CARS are not posttranslational modifiers of cysteine residues, but can co-translationally affect protein synthesis through the generation of persulfidated cysteinyl-tRNA and subsequent incorporation of cysteine persulfide and polysulfides into the nascent polypeptide chain during translation (Akaike et al. 2017). The function and stability of persulfidated cysteine residues in the native polypeptide chain, however, remains elusive.

3.4 D-Cysteine desulfhydrase

Cysteine desulfhydrases (CDES) are PLP-dependent enzymes that decompose cysteine to produce pyruvate, ammonium, and H₂S. Cysteine desulfhydrases are widely conserved in bacteria and plants and were shown to use L-cysteine (L-cysteine desulfhydrases; LCDES; EC 4.4.1.28) and/or D-cysteine (D-cysteine desulfhydrases; DCDES; EC 4.4.1.15) as substrates (Nagasawa et al. 1985; Riemenschneider et al. 2005b; Schmidt 1982; Todorovic and Glick 2008). The Arabidopsis genome encodes at least four proteins with reported CDES activity: LCD (At3g62130) and DES1 (At5g28030) that are thought to reside in the cytosol and to exclusively accept L-cysteine as substrate (Álvarez et al. 2010; Shen et al. 2012) and DCDES1 (At1g48420) and DCDES2 (At3g26115) that accept D-cysteine as substrate (Riemenschneider et al. 2005a). While the subcellular localization of DCDES2 has not been experimentally determined, several lines of evidence support DCDES1 mitochondrial localization (Riemenschneider et al. 2005b; Senkler et al. 2017; Fuchs et al. 2020). DCDES1 thus likely contributes to mitochondrial H₂S production. Indeed, Arabidopsis dcdes1 null mutants display a significantly decreased H₂S level (Li et al. 2018). Furthermore, alterations of the DCDES1 transcript level result in phenotypes that were also linked with exogenous H₂S application on plants, including cadmium tolerance and fruit ripening (Jia et al. 2016; Mostofa et al. 2015; Yao et al. 2018; Zhang et al. 2020; Zhao et al. 2023). Growth of Arabidopsis seedlings on cadmium can induce DCDES1 expression and elevate the total H₂S production rate (Zhang et al. 2020), effects that were previously also shown for the cytosolic DES1 (Jia et al. 2016). Similarly, constitutive heterologous expression of Arabidopsis DCDES1 in E. coli lead to increased H₂S levels and enhanced cadmium tolerance (Shen et al. 2012). Notably, Arabidopsis DCDES1 can apparently be persulfidated (Jurado-Flores et al. 2023), allowing to speculate about the integration of DCDES1 in regulatory loops as it was shown for DES1 (Shen et al. 2020). Worth mentioning is that it was shown that elevated H₂S levels improved plant growth under Cd-stress by reduction of cadmium-induced oxidative stress in rice and alfalfa (Cui et al. 2014; Mostofa et al. 2015). In rice plants suffering Cd-stress, O₂^·− and H₂O₂ levels were reduced when treated with NaHS (Mostofa et al. 2015). Considering the protective effects of persulfides as mentioned above (2.1. Protective effect of persulfides by enhanced oxidant scavenging) this could explain these results although they have to be confirmed for the mitochondrial compartment.

Despite these correlative observations, our understanding of D-cysteine’s physiological importance and the mechanistic links between increased H₂S levels and the observed phenotypes remains largely limited. While conservation of DCDES genes across the green linage argues for a functional significance, the source of D-cysteine, as substrate to produce H₂S, is unclear. In mammals, D-cysteine was suggested to be endogenously produced by serine racemase (SR), although recombinantly produced mammalian SR showed no measurable L- to D-cysteine conversion activity in vitro (Semenza et al. 2021; Shibuya et al. 2013). Arabidopsis possesses an SR homolog (At4g11640), but no L- to D-cysteine racemase activity has been demonstrated yet. While other D-amino acids, such as D-alanine, were shown to be present in soil and to be taken up directly by Arabidopsis, no such uptake data has been described for D-cysteine (Brodowski et al. 2005; Gördes et al. 2011).

4.1 Depersulfidation of proteins via cellular reducing systems

To harness persulfidation for the dynamic switching of protein behavior and characteristics, cells must also be able to selectively depersulfidate proteins. In mammals, the thioredoxin (Trx)/thioredoxin reductase (TrxR) system was shown to play a major role in protein depersulfidation (Dóka et al. 2016; Wedmann et al. 2016) as mentioned above (Section 2.2. Protective effect of persulfidation). TrxR, in concert with human Trx1 and the Thioredoxin-related Protein of 14 kDa (TRP14), was shown to reduce bovine serum albumin persulfide (BSA-SSH) in vitro and chemical/genetic interference with the Trx/TrxR system led to global increases of persulfidation levels in human cells (Dóka et al. 2016; Wedmann et al. 2016). Mammalian selenocysteine (Sec)-TrxR itself exhibits polysulfide-reducing activity in vitro that is improved by the presence of Trxs (Dóka et al. 2016). In contrast, Arabidopsis NADPH-dependent thioredoxin reductase NTRB, which does not possess a selenocysteine residue, has no depersulfidation activity (Moseler et al. 2021). Whether plant TRXs also regulate cellular persulfidation by restoring reduced thiols has not yet been tested. At least in vitro, cytosolic TRXh1 and mitochondrial TRXo1 and TRXo2 were able to interact with MSTs to release H₂S and restore the activity of persulfidated MSTs after oxidative stress by H₂O₂ treatment (Moseler et al. 2021). Canonical Trxs are characterized by the presence of two cysteine residues in a CxxC motif in their active site, both of them being indispensable for their disulfide and also persulfide reducing activity. Accordingly, a decreased H₂S release was observed in the presence of Trx variants mutated for either cysteines (Moseler et al. 2021). Trx exerts its depersulfidation activity through two possible mechanisms (Libiad et al. 2018; Vignane and Filipovic 2023). The first possibility is that the outer sulfur of the persulfide is transferred in a transpersulfidation reaction to the catalytic cysteine of the Trx to transiently form Trx-SSH. Trx-SSH is then attacked by the resolving cysteine of Trx to displace HS⁻ and form an intramolecular disulfide bond within Trx. Alternatively, one of the two Trx cysteines could directly attack the inner persulfide sulfur to eliminate H₂S and form a mixed Trx-target disulfide. The Trx-target disulfide would then further react with the resolving cysteine of Trx to substitute the target and form an intramolecular disulfide bond within Trx. Because thiol-disulfide exchange reactions occur through a 180° attack of the free thiol on a disulfide, it can be assumed the mechanism through which the reaction proceeds is determined by steric hindrance (Lillig and Berndt 2013). In human cells, it was shown that Trx1 forms mixed disulfides with pyruvate carboxylase and GAPDH indicating a preference for the second mechanism with an attack on the inner sulfur atom (Ju et al. 2016). Also, the glutaredoxin (GRX) system including a class I GRX, GSH, glutathione reductase and NADPH is able to reduce protein persulfides (Dóka et al. 2016; Moseler et al. 2021). Similar to TRXs, class I GRXs contain two cysteines in their active site. In the reaction mechanism, however, only the catalytic cysteine is required (Moseler et al. 2021). In plant mitochondria, no class I GRX is present excluding therefore this mechanism of protein depersulfidation (Schlößer et al. 2024). Importantly, independent of the reducing system involved, protein depersulfidation can generate free sulfide.

4.2 Detoxification of sulfide

Sulfide is a potent, non-competitive, reversible inhibitor of cytochrome c oxidase (COX; syn. complex IV) in the mitochondrial ETC. It binds to the oxidized states of the binuclear center of COX and the inhibited state is assumed to have one sulfide bound to the reduced copper ion Cu_B and the other bound to oxidized heme a₃ (Hill et al. 1984; Nicholls et al. 2013). Sulfide inhibits Arabidopsis COX efficiently at low nanomolar concentrations (IC₅₀ = 6.9 nM; (Birke et al. 2012)) and animal COX at higher nanomolar concentrations (e.g. IC₅₀ = 130 nM; K_i = 200 nM (Petersen 1977; Roth et al. 1998)). Animal and plant mitochondria thus require sulfide detoxification systems to maintain ATP production by the ETC. These systems help to keep sulfide levels in animal tissues in the low nanomolar range (Furne et al. 2008).

In fission yeast and animals, sulfide is consumed by the inner mitochondrial membrane-bound sulfide:quinone oxidoreductase (SQR) (Hildebrandt and Grieshaber 2008; Shahak and Hauska 2008). SQRs are additionally found in many bacteria and were, from an evolutionary perspective, essential for early life forms in the pre-oxygenic world where sulfide served as an energy source (Olson and Straub 2015). SQRs catalyze two half-reactions: transfer of electrons to the quinone pool of the ETC to ultimately reduce oxygen and sulfur transfer to an thiophilic acceptor, primarily GSH with GSSH as product (Figure 5A) (Landry et al. 2017, 2019, Libiad et al. 2014; Jackson et al. 2019). GSSH has been reported to be exported from the mitochondrial matrix via the ATP-binding cassette transporter ATM3 in the inner mitochondrial membrane (Schaedler et al. 2014). Alternatively, GSSH is consumed by persulfide dioxygenase ETHE1 in the mitochondrial matrix which produces sulfite. The resulting sulfite gets either oxidized to sulfate by sulfite oxidase (SO) or metabolized to thiosulfate by the thiosulfate sulfurtransferase (TST; syn. rhodanese) (Filipovic et al. 2018). Notably, in mice and human cells up to 1 % of the GSH pool is persulfidated indicating that GSSH is not just a side product (Barayeu et al. 2023b; Ida et al. 2014). It has been suggested that GSSH has cytoprotective effects and can scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis (Barayeu et al. 2023b).

Figure 5:

Key sulfide detoxification mechanisms in animals and plants. (A) In animals, the sulfide:quinone reductase (SQR) at the inner mitochondrial membrane transfers the electrons from sulfides to the quinone (coenzyme Q; CoQ) pool and the sulfur to reduced glutathione (GSH) to produce glutathione persulfide (GSSH). GSSH is consumed by oxygen-dependent ETHE1 with sulfite and glutathione as product. Sulfite is either oxidized to sulfate by sulfite oxidase (SO) or metabolized to thiosulfate by the thiosulfate sulfurtransferase (TST; syn. rhodanese). III: complex III of the mitochondrial electron transport chain. (B) In plants, sulfide is used to synthesize cysteine through the cysteine synthase complex (CSC). Serine acetyltransferase (SERAT) produces the cysteine precursor O-acetylserine (OAS) from serine and acetyl coenzyme A and O-acetylserine (thiol) lyase (OAS-TL) incorporates sulfide into OAS to form cysteine.

Plants lack SQR and rhodanese homologs (Table 2) and thus require alternative routes for sulfide detoxification (Moseler et al. 2020; Theissen et al. 2003). Although plants contain homologs of ETHE1, it is unclear whether plant ETHE1, in the absence of SQR, substantially contributes to consumption of sulfide. Efficient removal of sulfide through plant ETHE1 would require that sulfide efficiently forms a persulfide with oxidized glutathione (GSSG) in a first step. The embryo-lethality of an ethe1 knock-out, and the severe embryo-developmental effects of an ethe1-1 knock-down, are accordingly thought to be associated with the role of ETHE1 in cysteine catabolism (Holdorf et al. 2012; Höfler et al. 2016; Krüßel et al. 2014). The key mechanism that plants use to detoxify mitochondrial sulfide is thought to be the mitochondrial matrix-localized cysteine synthase complex (CSC). The CSC consists of serine acetyltransferase2;2 (SERAT2;2) and O-acetylserine (thiol) lyase C (OAS-TL C) (Álvarez et al. 2012b; Birke et al. 2015; Wirtz et al. 2010). SERAT produces the cysteine precursor O-acetylserine (OAS) from serine and acetyl coenzyme A and OAS-TL incorporates sulfide into OAS to form cysteine (Figure 5B). Arabidopsis possesses two further OAS-TL isoforms, A and B, that were shown to reside in the cytosol and plastids, respectively (reviewed in Hell et al. (2002)). It is, however, unlikely that they contribute a major detoxification capacity for mitochondrial sulfide since sulfide predominantly exists as charged hydrosulfide anion (HS⁻) in the alkaline mitochondrial matrix and can as such not freely permeate across the mitochondrial membrane. Although mitochondrial OAS-TL C contributes only little to net cysteine synthesis of the plant cell, oas-tl c null mutants suffer from inhibition of COX activity (Birke et al. 2012), demonstrating the role of OAS-TL C in clearance of toxic sulfide. Notably, the affinity of Arabidopsis OAS-TL C for sulfide is in the low micromolar range (Wirtz et al. 2004) and thus much higher than the IC₅₀ of COX for sulfide. Efficient removal of sulfide from plant mitochondria may thus require additional detoxification systems. Recently, the cytosolic superoxide dismutase SOD1 from yeast was shown to contribute to sulfide detoxification (Switzer et al. 2023). Plants contain mitochondrial homologs of yeast SOD1, but their role in sulfide clearance is currently unclear.