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• W3129192891 abstract "The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form. The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form. An ever-growing body of evidence indicates that hydrogen sulfide (H2S) plays a role in cellular signaling as other gaseous molecules such as nitric oxide (NO) and carbon monoxide (CO). Signaling by H2S is proposed to occur via the posttranslational modification (PTM) of critical cysteine residues (RSH) to persulfides (RSSH), called persulfidation, resulting in a cysteine whose thiol group is covalently bound to sulfur (sulfane sulfur) (1Hanaoka K. Sasakura K. Suwanai Y. Toma-Fukai S. Shimamoto K. Takano Y. Shibuya N. Terai T. Komatsu T. Ueno T. Ogasawara Y. Tsuchiya Y. Watanabe Y. Kimura H. Wang C. et al.Discovery and mechanistic characterization of selective inhibitors of H2S-producing enzyme: 3-mercaptopyruvate sulfurtransferase (3MST) targeting active-site cysteine persulfide.Sci. Rep. 2017; 7: 40227Crossref PubMed Scopus (49) Google Scholar). Oxidized thiol species such as sulfenic acids (RSOH), but not reduced thiols, are the direct targets of H2S reactivity (2Francoleon N.E. Carrington S.J. Fukuto J.M. The reaction of H2S with oxidized thiols: Generation of persulfides and implications to H2S biology.Arch. Biochem. Biophys. 2011; 516: 146-153Crossref PubMed Scopus (144) Google Scholar, 3Wedmann R. Onderka C. Wei S. Szijártó I.A. Miljkovic J.L. Mitrovic A. Lange M. Savitsky S. Yadav P.K. Torregrossa R. Harrer E.G. Harrer T. Ishii I. Gollasch M. Wood M.E. et al.Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation.Chem. Sci. 2016; 7: 3414-3426Crossref PubMed Google Scholar, 4Zivanovic J. Kouroussis E. Kohl J.B. Adhikari B. Bursac B. Schott-Roux S. Petrovic D. Miljkovic J.L. Thomas-Lopez D. Jung Y. Miler M. Mitchell S. Milosevic V. Gomes J.E. Benhar M. et al.Selective persulfide detection reveals evolutionarily conserved antiaging effects of S-sulfhydration.Cell Metab. 2020; 31: 1152-1170.e13Abstract Full Text Full Text PDF Scopus (8) Google Scholar). Cysteine persulfides have been found in free cysteine, small molecule peptides, as well as in proteins (5Millikin R. Bianco C.L. White C. Saund S.S. Henriquez S. Sosa V. Akaike T. Kumagai Y. Soeda S. Toscano J.P. Lin J. Fukuto J.M. The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation.Free Radic. Biol. Med. 2016; 97: 136-147Crossref PubMed Scopus (63) Google Scholar). Recently, it was demonstrated that prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs) have a crucial role in the synthesis of cysteine persulfides (Cys-SSH) (6Akaike T. Ida T. Wei F.-Y. Nishida M. Kumagai Y. Alam M.M. Ihara H. Sawa T. Matsunaga T. Kasamatsu S. Nishimura A. Morita M. Tomizawa K. Nishimura A. Watanabe S. et al.Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics.Nat. Commun. 2017; 8: 1-15Crossref PubMed Scopus (221) Google Scholar, 7Fujii S. Sawa T. Motohashi H. Akaike T. Persulfide synthases that are functionally coupled with translation mediate sulfur respiration in mammalian cells.Br. J. Pharmacol. 2019; 176: 607-615Crossref PubMed Scopus (22) Google Scholar) thus representing one of the numerous pathways contributing to the formation of persulfides. In plants, H2S is associated with various physiological functions ranging from responses to abiotic and biotic stresses, plant development (seed germination, root development, leaf senescence), photosynthesis, and autophagy to stomatal movement (8Gotor C. García I. Aroca Á. Laureano-Marín A.M. Arenas-Alfonseca L. Jurado-Flores A. Moreno I. Romero L.C. Signaling by hydrogen sulfide and cyanide through post-translational modification.J. Exp. Bot. 2019; 70: 4251-4265Crossref PubMed Scopus (49) Google Scholar). Three distinct enzymatic pathways producing H2S have been identified. Sulfide is primarily produced in chloroplasts through the action of sulfite reductase during the reductive assimilation of sulfate. It is then incorporated into the amino acid skeleton of O-acetylserine to form cysteine, the biosynthesis of which can occur in the cytosol, plastids, and mitochondria (9Takahashi H. Kopriva S. Giordano M. Saito K. Hell R. Sulfur assimilation in photosynthetic organisms: Molecular functions and regulations of transporters and assimilatory enzymes.Annu. Rev. Plant Biol. 2011; 62: 157-184Crossref PubMed Scopus (493) Google Scholar). Another pathway of H2S biogenesis is the conversion of cyanide and cysteine into β-cyanoalanine and H2S, which is catalyzed by the β-cyanoalanine synthase CAS-C1 in mitochondria (8Gotor C. García I. Aroca Á. Laureano-Marín A.M. Arenas-Alfonseca L. Jurado-Flores A. Moreno I. Romero L.C. Signaling by hydrogen sulfide and cyanide through post-translational modification.J. Exp. Bot. 2019; 70: 4251-4265Crossref PubMed Scopus (49) Google Scholar, 10Yamaguchi Y. Nakamura T. Kusano T. Sano H. Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and β-cyanoalanine synthase.Plant Cell Physiol. 2000; 41: 465-476Crossref PubMed Scopus (76) Google Scholar). In the cytosol, the L-cysteine desulfhydrase 1 (DES1) degrades cysteine into H2S, ammonia, and pyruvate (11Álvarez C. Calo L. Romero L.C. García I. Gotor C. An acetylserine(thiol)lyase homolog with cysteine desulfhydrase activity regulates cysteine homeostasis in Arabidopsis.Plant Physiol. 2010; 152: 656-669Crossref PubMed Scopus (242) Google Scholar, 12Álvarez C. García I. Moreno I. Pérez-Pérez M.E. Crespo J.L. Romero L.C. Gotor C. Cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile in Arabidopsis.Plant Cell. 2012; 24: 4621-4634Crossref PubMed Scopus (120) Google Scholar). Up to now, the links between the H2S-producing enzymes and the cellular persulfidation state have not been clearly identified in plants. Although des1 mutant plants display a reduced sulfide production (30% decrease under steady-state growth conditions) (11Álvarez C. Calo L. Romero L.C. García I. Gotor C. An acetylserine(thiol)lyase homolog with cysteine desulfhydrase activity regulates cysteine homeostasis in Arabidopsis.Plant Physiol. 2010; 152: 656-669Crossref PubMed Scopus (242) Google Scholar) and are affected in several physiological pathways (senescence, autophagy, stomatal closure, and immunity), the number of persulfidated proteins in wildtype (WT) and des1 plants (2015 and 2130, respectively) is similar and with a high overlap of 85% (13Aroca A. Benito J.M. Gotor C. Romero L.C. Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis.J. Exp. Bot. 2017; 68: 4915-4927Crossref PubMed Scopus (126) Google Scholar). In des1 mutants, the persulfidation level of only 47 proteins, including protein kinases, protein phosphatases, and abscisic acid receptors, was decreased underlying a limited role of DES1 in protein persulfidation. Hence, other factors/pathways promoting protein persulfidation in plants remain to be identified. In mammals, H2S is generated primarily by three different enzymes: cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfurtransferase (MST) (14Rose P. Moore P.K. Zhu Y.Z. H2S biosynthesis and catabolism: New insights from molecular studies.Cell. Mol. Life Sci. 2017; 74: 1391-1412Crossref PubMed Scopus (83) Google Scholar, 15Filipovic M.R. Zivanovic J. Alvarez B. Banerjee R. Chemical biology of H2S signaling through persulfidation.Chem. Rev. 2018; 118: 1253-1337Crossref PubMed Scopus (351) Google Scholar). MSTs belong to the sulfurtransferase (STR) family, which are characterized by the presence of a rhodanese (Rhd) domain (16Bordo D. Bork P. The rhodanese/Cdc25 phosphatase superfamily.EMBO Rep. 2002; 3: 741-746Crossref PubMed Scopus (257) Google Scholar, 17Moseler A. Selles B. Rouhier N. Couturier J. Novel insights into the diversity of the sulfurtransferase family in photosynthetic organisms with emphasis on oak.New Phytol. 2020; 226: 967-977Crossref PubMed Scopus (10) Google Scholar). Owing to the conserved catalytic cysteine present in the rhodanese domain, STRs are implicated in sulfur/persulfide trafficking through their ability to catalyze the transfer of a sulfur atom to nucleophilic acceptors. The MST isoforms are characterized by the presence of two Rhd domains with only the C-terminal one possessing the catalytic cysteine in a characteristic CG[S/T]GVT signature (17Moseler A. Selles B. Rouhier N. Couturier J. Novel insights into the diversity of the sulfurtransferase family in photosynthetic organisms with emphasis on oak.New Phytol. 2020; 226: 967-977Crossref PubMed Scopus (10) Google Scholar). In mammals, MSTs are found in both the cytosol and mitochondria. In rat liver, the specific MST activity is 3-fold higher in mitochondria than in the cytosol (18Nagahara N. Ito T. Kitamura H. Nishino T. Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: Confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis.Histochem. Cell Biol. 1998; 110: 243-250Crossref PubMed Scopus (167) Google Scholar). In mice, the mitochondrial MST contributes to H2S metabolism and sulfide signaling by releasing H2S in the presence of a reductant such as a thioredoxin (TRX) (19Shibuya N. Tanaka M. Yoshida M. Ogasawara Y. Togawa T. Ishii K. Kimura H. 3-mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane sulfur in the brain.Antioxid. Redox Signal. 2008; 11: 703-714Crossref Scopus (703) Google Scholar, 20Mikami Y. Shibuya N. Kimura Y. Nagahara N. Ogasawara Y. Kimura H. Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide.Biochem. J. 2011; 439: 479-485Crossref PubMed Scopus (204) Google Scholar). Furthermore, the production of polysulfides H2S2 and H2S3 by MST has been reported in the absence of reductant (21Kimura Y. Toyofuku Y. Koike S. Shibuya N. Nagahara N. Lefer D. Ogasawara Y. Kimura H. Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.Sci. Rep. 2015; 5: 14774Crossref PubMed Scopus (147) Google Scholar), whereas Cys-SSH and glutathione persulfide (GSSH) were observed in the presence of physiological concentrations of cysteine and GSH (22Kimura Y. Koike S. Shibuya N. Lefer D. Ogasawara Y. Kimura H. 3-mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H2S2, H2S3 and H2S.Sci. Rep. 2017; 7: 10459Crossref PubMed Scopus (74) Google Scholar). Similar results were observed in Escherichia coli with the involvement of the MST ortholog, SseA, in the production of reactive sulfane sulfur and notably GSSH and GSSSH (23Li K. Xin Y. Xuan G. Zhao R. Liu H. Xia Y. Xun L. Escherichia coli uses separate enzymes to produce H2S and reactive sulfane sulfur from L-cysteine.Front. Microbiol. 2019; 10: 298Crossref PubMed Scopus (23) Google Scholar). From a physiological point of view, the levels of total persulfidated species in the brain of MST-KO mice are less than 50% of those in the brain of WT mice indicating that mitochondrial MST is indeed an important factor in promoting protein persulfidation (21Kimura Y. Toyofuku Y. Koike S. Shibuya N. Nagahara N. Lefer D. Ogasawara Y. Kimura H. Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.Sci. Rep. 2015; 5: 14774Crossref PubMed Scopus (147) Google Scholar, 22Kimura Y. Koike S. Shibuya N. Lefer D. Ogasawara Y. Kimura H. 3-mercaptopyruvate sulfurtransferase produces potential redox regulators cysteine- and glutathione-persulfide (Cys-SSH and GSSH) together with signaling molecules H2S2, H2S3 and H2S.Sci. Rep. 2017; 7: 10459Crossref PubMed Scopus (74) Google Scholar). The biochemical characterization of human MSTs has confirmed that TRXs are physiological persulfide acceptors contributing to the generation of H2S and thus TRXs can be considered as regulators of protein persulfide levels in the cells (24Yadav P.K. Vitvitsky V. Carballal S. Seravalli J. Banerjee R. Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically relevant concentrations.J. Biol. Chem. 2020; 295: 6299-6311Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although good evidence for the function of MSTs in sulfur transfer or H2S synthesis has been gained in bacteria and vertebrates over the last decade, their function in plants is just being elucidated. Plants possess at least one MST isoform, but Arabidopsis thaliana has two MSTs, named STR1 and STR2, which are located in mitochondria and cytosol, respectively (25Bauer M. Dietrich C. Nowak K. Sierralta W.D. Papenbrock J. Intracellular localization of Arabidopsis sulfurtransferases.Plant Physiol. 2004; 135: 916-926Crossref PubMed Scopus (32) Google Scholar, 26Nakamura T. Yamaguchi Y. Sano H. Plant mercaptopyruvate sulfurtransferases.Eur. J. Biochem. 2000; 267: 5621-5630Crossref PubMed Scopus (50) Google Scholar). In Arabidopsis, the MSTs are suggested to be multifunctional enzymes involved in cysteine catabolism and sulfide production and possibly in cyanide detoxification as shown in vitro (27Krüßel L. Junemann J. Wirtz M. Birke H. Thornton J.D. Browning L.W. Poschet G. Hell R. Balk J. Braun H.-P. Hildebrandt T.M. The mitochondrial sulfur dioxygenase ethylmalonic encephalopathy protein1 is required for amino acid catabolism during carbohydrate starvation and embryo development in Arabidopsis.Plant Physiol. 2014; 165: 92-104Crossref PubMed Scopus (40) Google Scholar, 28Höfler S. Lorenz C. Busch T. Brinkkötter M. Tohge T. Fernie A.R. Braun H.-P. Hildebrandt T.M. Dealing with the sulfur part of cysteine: Four enzymatic steps degrade L-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.Physiol. Plant. 2016; 157: 352-366Crossref PubMed Scopus (12) Google Scholar, 29Mao G. Wang R. Guan Y. Liu Y. Zhang S. Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana.J. Biol. Chem. 2011; 286: 7548-7557Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30Papenbrock J. Schmidt A. Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana.Eur. J. Biochem. 2000; 267: 5571-5579Crossref PubMed Scopus (46) Google Scholar). Regarding the MST role in cysteine degradation in mitochondria, STR1 converts 3-MP, formed by the action of a yet unknown cysteine aminotransferase on cysteine, to pyruvate resulting in the formation of an enzyme-bound persulfide (Fig. S1). It is suggested that STR1 persulfide is then transferred to GSH (28Höfler S. Lorenz C. Busch T. Brinkkötter M. Tohge T. Fernie A.R. Braun H.-P. Hildebrandt T.M. Dealing with the sulfur part of cysteine: Four enzymatic steps degrade L-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.Physiol. Plant. 2016; 157: 352-366Crossref PubMed Scopus (12) Google Scholar). In accordance with their catalytic mechanism, both Arabidopsis MSTs were isolated as persulfidated proteins from leaf extracts (13Aroca A. Benito J.M. Gotor C. Romero L.C. Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis.J. Exp. Bot. 2017; 68: 4915-4927Crossref PubMed Scopus (126) Google Scholar). In addition to these functions, an interaction of both MSTs was observed with TRXs through bimolecular fluorescence complementation (BiFC), but no further investigation of the implication of these two systems in H2S biosynthesis and protein persulfidation has been performed (31Henne M. König N. Triulzi T. Baroni S. Forlani F. Scheibe R. Papenbrock J. Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.FEBS Open Bio. 2015; 5: 832-843Crossref PubMed Scopus (7) Google Scholar). In this study, we have investigated the kinetics of H2S biosynthesis and of low-molecular-weight persulfide production from 3-MP catalyzed by recombinant Arabidopsis STR1 and STR2 since a fine characterization of their enzymatic properties was not achieved so far. This 3-MP conversion constitutes an efficient H2S biogenesis system in the presence of the major cellular reductants (TRX, GRX). Other trans-persulfidation reactions, not necessarily releasing H2S, have been observed with GSH, Cys, or the model acceptor protein roGFP2, suggesting that MSTs could participate in the trafficking of sulfane sulfur and/or in protein oxidation. These results advance our understanding of the roles of these two MSTs. The observed partnership with physiological sulfur acceptors such as TRXs, GRXs, and GSH will help mapping sulfur transfer events across interconnected pathways and designing adequate strategies for studying H2S biogenesis in planta. To analyze the functional facets of Arabidopsis MSTs, the corresponding recombinant proteins were purified after heterologous expression in E. coli with a production yield for STR1 of 20 mg and for STR2 of 4 mg of protein from 1 l bacterial culture. Different sulfur-containing compounds were tested as substrates to examine which sulfur donor is preferentially used by STR1 and STR2 in vitro. The protein activity was quantified by measuring the DTT-released H2S through the formation of methylene blue (Fig. S2) (32Hartle M.D. Pluth M.D. A practical guide to working with H2S at the interface of chemistry and biology.Chem. Soc. Rev. 2016; 45: 6108-6117Crossref PubMed Google Scholar). The data obtained clearly indicated that 3-MP was the preferred sulfur donor that leads to H2S production (Fig. 1A). Only a very low activity was observed with thiosulfate as sulfur donor (Fig. 1A), although it was shown previously that STR1 possessed a thiosulfate:cyanide sulfurtransferase activity (33Burow M. Kessler D. Papenbrock J. Enzymatic activity of the Arabidopsis sulfurtransferase resides in the C-terminal domain but is boosted by the N-terminal domain and the linker peptide in the full- length enzyme.Biol. Chem. 2002; 383: 1363-1372Crossref PubMed Scopus (10) Google Scholar). No activity was measured in the presence of cysteine. The interaction of 3-MP with both STR1 and STR2 was then evaluated using fluorescence measurements. Indeed, some non-plant MSTs exhibit quenched intrinsic fluorescence after forming the intermediate persulfide, owing to the location of a tryptophan residue close to the active site (34Colnaghi R. Cassinelli G. Drummond M. Forlani F. Pagani S. Properties of the Escherichia coli rhodanese-like protein SseA: Contribution of the active-site residue Ser240 to sulfur donor recognition.FEBS Lett. 2001; 500: 153-156Crossref PubMed Scopus (24) Google Scholar, 35Fräsdorf B. Radon C. Leimkühler S. Characterization and interaction studies of two isoforms of the dual localized 3-mercaptopyruvate sulfurtransferase TUM1 from humans.J. Biol. Chem. 2014; 289: 34543-34556Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 36Lec J.-C. Boutserin S. Mazon H. Mulliert G. Boschi-Muller S. Talfournier F. Unraveling the mechanism of cysteine persulfide formation catalyzed by 3-mercaptopyruvate sulfurtransferases.ACS Catal. 2018; 8: 2049-2059Crossref Scopus (9) Google Scholar). Similar to E. coli SseA or human TUM1, STR1 and STR2 display intrinsic fluorescence with a maximum at 336 nm when excited at 270 nm (Fig. S3A). The fluorescence emission spectra of either Arabidopsis MSTs did not change following the addition of pyruvate but showed a strong decrease after addition of 3-MP (Fig. S3, B–D). Thus, we analyzed the fluorescence changes as a function of 3-MP concentration to determine the STR dissociation constant for 3-MP. Kd values of 1.3 ± 0.9 and 0.7 ± 0.3 μM were obtained for STR1 and STR2, respectively (Fig. 1B). In addition, STR1 C333S and STR2 C298S variants, in which the catalytically important cysteine residue present in the 6-amino acid CGTGVT signature was substituted for a serine, were generated. The intrinsic fluorescence of both variants also decreased after addition of 3-MP but not as strongly as in STR1 and STR2 indicating that the binding of 3-MP already led to fluorescence quenching, not only persulfide formation (Fig. S3, B–D). Then we examined whether MSTs are able to transfer a persulfide from 3-MP to an acceptor protein. Therefore, we used the redox-sensitive green fluorescent protein (roGFP2) as a model acceptor protein to analyze protein trans-persulfidation in vitro based on a previous work in which it was observed that roGFP2 can be oxidized through the activity of MSTs using 3-MP as sulfur donor in living cells (37Ezeriņa D. Takano Y. Hanaoka K. Urano Y. Dick T.P. N-acetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur production.Cell Chem. Biol. 2018; 25: 447-459Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Considering that 3-MP alone does not trigger roGFP2 oxidation and that the reaction between 3-MP and MSTs leads to the persulfidation of the MST catalytic cysteine, it is likely that this persulfide group is then transferred to one of the Cys of the reduced roGFP2 and that the second Cys of roGFP2 reduces the persulfide, yielding H2S and an oxidized roGFP2 (Fig. 2A). The resulting intramolecular disulfide bridge of roGFP2 changes the steric arrangement of the beta barrel surface and in turn the optical characteristics of the chromophore enabling a direct readout of roGFP2 oxidation. Addition of 3-MP to the MST and roGFP2 mix led indeed to an efficient oxidation of roGFP2, whereas no oxidation was observed in the absence of MST or using variants mutated for the conserved active site Cys of the MST (STR1 C333S and STR2 C298S) (Fig. 2, B and C). Altogether, these findings indicate that STR1 and STR2 form persulfides on the catalytic cysteine after treatment with 3-MP, which is the canonical substrate in vitro and is able to transfer the persulfide to an acceptor. Earlier it was suggested that STR1 plays a role in cysteine degradation by transferring a persulfide from 3-MP to GSH (28Höfler S. Lorenz C. Busch T. Brinkkötter M. Tohge T. Fernie A.R. Braun H.-P. Hildebrandt T.M. Dealing with the sulfur part of cysteine: Four enzymatic steps degrade L-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.Physiol. Plant. 2016; 157: 352-366Crossref PubMed Scopus (12) Google Scholar), and in addition, it was reported through BiFC that STR1 and STR2 interact with the mitochondrial TRXo1 or cytosolic TRXh1, respectively (31Henne M. König N. Triulzi T. Baroni S. Forlani F. Scheibe R. Papenbrock J. Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.FEBS Open Bio. 2015; 5: 832-843Crossref PubMed Scopus (7) Google Scholar). These results prompted us to investigate the interaction of the MSTs with a larger panel of physiologically relevant sulfane sulfur acceptors, GSH and cysteine, and also TRX or GRX reducing systems and to determine the steady-state kinetic parameters associated with H2S generation (Table 1). In all cases, a hyperbolic relationship between the rate of reaction and the concentration of acceptor was obtained (Fig. S4, A–E). Catalytic efficiencies (kcat/Km) ranging from 5.2 × 105 to 8.7 × 106 M−1 s−1 have been measured in the presence of the TRX or GRX reducing systems for both STR1 and STR2, which are 10- to 100-fold higher than the values obtained with GSH or cysteine (Table 1). The apparent Km values for the two mitochondrial TRXo1 and TRXo2 as well as the cytosolic TRXh1 were in the low micromolar range ranging from 1.3 to 5.3 μM. Similar apparent Km values around 3 μM were recently estimated for the human MPST1 and MPST2 with the cytosolic thioredoxin TXN (24Yadav P.K. Vitvitsky V. Carballal S. Seravalli J. Banerjee R. Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically relevant concentrations.J. Biol. Chem. 2020; 295: 6299-6311Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In contrast, the apparent Km values of STR1 or STR2 for GSH, 200 or 350 μM respectively, differ considerably from the value of 28 mM obtained for human MPST2 (38Yadav P.K. Yamada K. Chiku T. Koutmos M. Banerjee R. Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.J. Biol. Chem. 2013; 288: 20002-20013Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Of interest, apparent Km values of 0.7 to 4.6 μM were measured for GRXs (GRXC1 and C4) using both STRs indicating that MST activity can be also coupled to these reductases. Similar steady-state kinetic parameters were determined for STR1 using TRXo1, GRXC1, or GSH by following the NADPH consumption instead of measuring the methylene blue formation (Fig. S5). Finally, the apparent Km values for 3-MP were estimated at saturating concentrations of each acceptor using mitochondrial TRXo1 and TRXo2 for STR1 and the cytosolic TRXh1 for STR2 and ranged from ∼290 to ∼460 μM (Table 2, Fig. S4F). Altogether, these results show that both Arabidopsis MST isoforms have globally similar kinetic constants for each substrate or reducing system tested. The apparent affinities indicate that all these interactions may be physiologically relevant and point for the first time to a possible role of GRXs. Moreover, GSH may have an important role as an acceptor of persulfides from plant MSTs, unlike human MPST.Table 1Kinetic parameters of 3-MP sulfurtransferase activity of Arabidopsis STR1 and STR2 using distinct sulfur acceptorsAcceptorSTR1STR2Km (μM)kcat (s−1)kcat/Km (M−1 s−1)Km (μM)kcat (s−1)kcat/Km (M−1 s−1)TRXo15.3 ± 1.06.81.3 × 106nananaTRXo21.3 ± 0.23.62.8 × 106nananaTRXh1nanana1.4 ± 0.21.41.0 × 106GRXC41.1 ± 0.14.64.2 × 1064.6 ± 0.62.45.2 × 105GRXC10.7 ± 0.16.18.7 × 1061.43 ± 0.33.12.2 × 106GSH200 ± 205.32.7 × 104350 ± 102.36.6 × 103Cysteine2200 ± 4003.91.7 × 1031300 ± 701.51.2 × 103na, not analyzed.Steady-state kinetic parameters were determined by varying the acceptor concentration at a saturating concentration of 3-MP (n = 3; means ± SD). Open table in a new tab Table 2Kinetic parameters of sulfurtransferase activity of Arabidopsis STR1 and STR2 for 3-MPAcceptorSTR1STR2Km (μM)kcat (s−1)kcat/Km (M−1 s−1)Km (μM)kcat (s−1)kcat/Km (M−1 s−1)TRXo1286 ± 513.54.7 ×" @default.
• W3129192891 created "2021-03-01" @default.
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• W3129192891 creator A5047053189 @default.
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• W3129192891 date "2021-01-01" @default.
• W3129192891 modified "2023-10-14" @default.
• W3129192891 title "Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems" @default.
• W3129192891 cites W1563176897 @default.
• W3129192891 cites W1585043560 @default.
• W3129192891 cites W1587229625 @default.
• W3129192891 cites W1861673379 @default.
• W3129192891 cites W1963482333 @default.
• W3129192891 cites W1970753749 @default.
• W3129192891 cites W1983793380 @default.
• W3129192891 cites W1997676478 @default.
• W3129192891 cites W1998861061 @default.
• W3129192891 cites W2017545901 @default.
• W3129192891 cites W2033017235 @default.
• W3129192891 cites W2034579940 @default.
• W3129192891 cites W2038600558 @default.
• W3129192891 cites W2040682707 @default.
• W3129192891 cites W2041867306 @default.
• W3129192891 cites W2054064167 @default.
• W3129192891 cites W2056306665 @default.
• W3129192891 cites W2060053190 @default.
• W3129192891 cites W2063207603 @default.
• W3129192891 cites W2072540192 @default.
• W3129192891 cites W2107395810 @default.
• W3129192891 cites W2107898534 @default.
• W3129192891 cites W2114076518 @default.
• W3129192891 cites W2118960787 @default.
• W3129192891 cites W2119726904 @default.
• W3129192891 cites W2121403476 @default.
• W3129192891 cites W2127347907 @default.
• W3129192891 cites W2133048919 @default.
• W3129192891 cites W2138481297 @default.
• W3129192891 cites W2140177413 @default.
• W3129192891 cites W2141576994 @default.
• W3129192891 cites W2151038773 @default.
• W3129192891 cites W2157677632 @default.
• W3129192891 cites W2160071206 @default.
• W3129192891 cites W2164287879 @default.
• W3129192891 cites W2167068434 @default.
• W3129192891 cites W2167641438 @default.
• W3129192891 cites W2167897698 @default.
• W3129192891 cites W2205995849 @default.
• W3129192891 cites W2218011606 @default.
• W3129192891 cites W2256535334 @default.
• W3129192891 cites W2272736396 @default.
• W3129192891 cites W2340296794 @default.
• W3129192891 cites W2364200124 @default.
• W3129192891 cites W2405848654 @default.
• W3129192891 cites W2409295593 @default.
• W3129192891 cites W2467056461 @default.
• W3129192891 cites W2552043470 @default.
• W3129192891 cites W2575515814 @default.
• W3129192891 cites W2748288120 @default.
• W3129192891 cites W2752688796 @default.
• W3129192891 cites W2766780446 @default.
• W3129192891 cites W2767914703 @default.
• W3129192891 cites W2785314193 @default.
• W3129192891 cites W2786677683 @default.
• W3129192891 cites W2801781332 @default.
• W3129192891 cites W2896362092 @default.
• W3129192891 cites W2916832354 @default.
• W3129192891 cites W2943218557 @default.
• W3129192891 cites W2944333101 @default.
• W3129192891 cites W2945242561 @default.
• W3129192891 cites W2965898235 @default.
• W3129192891 cites W2994885088 @default.
• W3129192891 cites W2998709095 @default.
• W3129192891 cites W3004729778 @default.
• W3129192891 cites W3009962493 @default.
• W3129192891 cites W3012102028 @default.
• W3129192891 cites W3032609687 @default.
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