Supplementary material for the article: Wedmann, R.; Onderka, C.; Wei, S.; Szijártó, I. A.; Miljkovic, J. L.; Mitrovic, A.; Lange, M.; Savitsky, S.; Yadav, P. K.; Torregrossa, R.; et al. Improved Tag-Switch Method Reveals That Thioredoxin Acts as Depersulfidase and Controls the Intracellular Levels of Protein Persulfidation. Chemical Science 2016, 7 (5), 3414–3426. https://doi.org/10.1039/c5sc04818d

Supplementary material for: [https://doi.org/10.1039/c5sc04818d]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1925

Insights into the mechanism of the reaction between hydrogen sulfide and peroxynitrite

Hydrogen sulfide and peroxynitrite are endogenously generated molecules that participate in biologically relevant pathways. A revision of the kinetic features of the reaction between peroxynitrite and hydrogen sulfide revealed a complex process. The rate constant of peroxynitrite decay, (6.65 ± 0.08) × 103 M-1 s-1 in 0.05 M sodium phosphate buffer (pH 7.4, 37 °C), was affected by the concentration of buffer. Theoretical modeling suggested that, as in the case of thiols, the reaction is initiated by the nucleophilic attack of HS- on the peroxide group of ONOOH by a typical bimolecular nucleophilic substitution, yielding HSOH and NO2 -. In contrast to thiols, the reaction then proceeds to the formation of distinct products that absorb near 408 nm. Experiments in the presence of scavengers and carbon dioxide showed that free radicals are unlikely to be involved in the formation of these products. The results are consistent with product formation involving the reactive intermediate HSSH and its fast reaction with a second peroxynitrite molecule. Mass spectrometry and UV-Vis absorption spectra predictions suggest that at least one of the products is HSNO2 or its isomer HSONO.Fil: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Carballal, Sebastián. Universidad de la República; UruguayFil: Wedmann, Rudolf. Universitat Erlangen-Nuremberg; AlemaniaFil: Morzan, Uriel N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Trujillo, Madia. Universidad de la República; UruguayFil: Radi, Rafael. Universidad de la República; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Filipovic, Milos R.. Universitat Erlangen-Nuremberg; AlemaniaFil: Alvarez, Beatriz. Universidad de la República; Urugua

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

Hydrogen sulfide (H₂S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H₂S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys–SSH) from cystine and H₂S from cysteine and/or homocysteine. Recently, Cys–SSH was proposed as the primary product of the transsulfuration pathway with H₂S representing a decomposition product of Cys–SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys–SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H₂S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H₂S rather than Cys–SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys–SSH product is short and decomposition leads to a mixture of polysulfides (Cys–S–(S)_<i>n</i>–S–Cys). These in vitro data, together with the intrinsic reactivity of Cys–SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions

Does Perthionitrite (SSNO^–) Account for Sustained Bioactivity of NO? A (Bio)chemical Characterization

Hydrogen sulfide (H₂S) and nitric oxide (NO) are important signaling molecules that regulate several physiological functions. Understanding the chemistry behind their interplay is important for explaining these functions. The reaction of H₂S with <i>S</i>-nitrosothiols to form the smallest <i>S</i>-nitrosothiol, thionitrous acid (HSNO), is one example of physiologically relevant cross-talk between H₂S and nitrogen species. Perthionitrite (SSNO^–) has recently been considered as an important biological source of NO that is far more stable and longer living than HSNO. In order to experimentally address this issue here, we prepared SSNO^– by two different approaches, which lead to two distinct species: SSNO^– and dithionitric acid [HON­(S)­S/HSN­(O)­S]. (H)­S₂NO species and their reactivity were studied by ¹⁵N NMR, IR, electron paramagnetic resonance and high-resolution electrospray ionization time-of-flight mass spectrometry, as well as by X-ray structure analysis and cyclic voltammetry. The obtained results pointed toward the inherent instability of SSNO^– in water solutions. SSNO^– decomposed readily in the presence of light, water, or acid, with concomitant formation of elemental sulfur and HNO. Furthermore, SSNO⁻ reacted with H₂S to generate HSNO. Computational studies on (H)­SSNO provided additional explanations for its instability. Thus, on the basis of our data, it seems to be less probable that SSNO^– can serve as a signaling molecule and biological source of NO. SSNO^– salts could, however, be used as fast generators of HNO in water solutions

Synthesis and Pharmacological Evaluation of Novel Adenine–Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H₂S slow-releasing moiety, coupled via a stable ether bond. The H₂S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives <b>4</b> and <b>11</b>, both containing 4-hydroxythiobenzamide moiety as H₂S donor, were selected for further in vivo evaluation. Their ability to release H₂S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection

Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation

Hydrogen sulfide (H S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or -sulfhydration) has been proposed as the main pathway for H S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H S release suggesting that thioredoxin could be an important regulator of H S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role

Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation

Hydrogen sulfide (H2S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or S-sulfhydration) has been proposed as the main pathway for H2S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H2S release suggesting that thioredoxin could be an important regulator of H2S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H2S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role in vivo.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3543