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

Beyond H₂S and NO Interplay: Hydrogen Sulfide and Nitroprusside React Directly to Give Nitroxyl (HNO). A New Pharmacological Source of HNO

Hydrogen sulfide (H₂S) has been increasingly recognized as an important signaling molecule that regulates both blood pressure and neuronal activity. Attention has been drawn to its interactions with another gasotransmitter, nitric oxide (NO). Here, we provide evidence that the physiological effects observed upon the application of sodium nitroprusside (SNP) and H₂S can be ascribed to the generation of nitroxyl (HNO), which is a direct product of the reaction between SNP and H₂S, not a consequence of released NO subsequently reacting with H₂S. Intracellular HNO formation has been confirmed, and the subsequent release of calcitonin gene-related peptide from a mouse heart has been demonstrated. Unlike with other thiols, SNP reacts with H₂S in the same way as rhodanese, i.e., the cyanide transforms into a thiocyanate. These findings shed new light on how H₂S is understood to interact with nitroprusside. Additionally, they offer a new and convenient pharmacological source of HNO for therapeutic purposes

Chemical Characterization of the Smallest <i>S</i>-Nitrosothiol, HSNO; Cellular Cross-talk of H₂S and <i>S</i>-Nitrosothiols

Dihydrogen sulfide recently emerged as a biological signaling molecule with important physiological roles and significant pharmacological potential. Chemically plausible explanations for its mechanisms of action have remained elusive, however. Here, we report that H₂S reacts with <i>S</i>-nitrosothiols to form thionitrous acid (HSNO), the smallest <i>S</i>-nitrosothiol. These results demonstrate that, at the cellular level, HSNO can be metabolized to afford NO⁺, NO, and NO^– species, all of which have distinct physiological consequences of their own. We further show that HSNO can freely diffuse through membranes, facilitating transnitrosation of proteins such as hemoglobin. The data presented in this study explain some of the physiological effects ascribed to H₂S, but, more broadly, introduce a new signaling molecule, HSNO, and suggest that it may play a key role in cellular redox regulation

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

Cytochrome <i>c</i> Reduction by H₂S Potentiates Sulfide Signaling

Hydrogen sulfide (H₂S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome <i>c</i> (Cyt C) by H₂S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H₂S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H₂S oxidized. H₂S increases O₂ consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H₂S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling

Nitric Oxide Is Reduced to HNO by Proton-Coupled Nucleophilic Attack by Ascorbate, Tyrosine, and Other Alcohols. A New Route to HNO in Biological Media?

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical

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