Reactive Sulfur Species Research Articles

Understanding Polysulfide-Mediated Papain Inhibition and Differentiating between Disulfide vs Persulfide Formation.

Protein cysteine residues are sensitive to redox-regulating molecules, including reactive sulfur species (RSS). As an important member of the RSS family, polysulfides are known to react with protein cysteines to form persulfides and disulfides, both affecting protein functions. In this work, we studied how polysulfides could impact cysteine proteases through careful mechanistic and kinetic studies. The model protein papain was treated with different polysulfides to elucidate the efficacy of polysulfides as inhibitors for this protein. We also explored the effects of different reductants that could regenerate papain activity after polysulfide-mediated inhibition. A triarylphosphine reagent, TXPTS, was found to be efficient in differentiating between papain persulfidation and disulfide formation.

Simultaneous Quantitation of Persulfides, Biothiols, and Hydrogen Sulfide through Sulfur Exchange Reaction with Trityl Spin Probes.

Reactive sulfur species (RSS) including persulfides (RSSHs), biothiols, and hydrogen sulfide (H2S) are key regulators in various physiological processes. To better understand the symbiotic relationship and interconversion of these RSS, it is highly desirable but challenging to develop analytical techniques that are capable of detecting and quantifying them. Herein, we report the rational design and synthesis of novel trityl-radical-based electron paramagnetic resonance (EPR) probes dubbed CT02-TNB and OX-TNB. CT02-TNB underwent fast sulfur exchange reactions with two reactive RSSHs (PS1 and PS2) which were released from their corresponding donors PSD1 and PSD2 to afford the specific conjugates. The resulting conjugates exhibit characteristic EPR spectra, thus enabling discriminative detection and quantitation of the two RSSHs. Moreover, CT02-TNB showed good response toward other RSS including glutathione (GSH), cysteine (Cys), H2S, and sulfite as well. Importantly, based on the updated EPR spectral simulation program, simultaneous quantitation of multiple RSS (e.g., PS1/GSH/Cys or PS1/GSH/H2S) by CT02-TNB was also achieved. Finally, the levels of released PS1 from PSD1 and endogenous GSH in isolated mouse livers were measured by the hydrophilic OX-TNB. This work represents the first study achieving discriminative and quantitative detection of different persulfides and other RSS by a spectroscopic method.

Exploring the Reactivity of Electrophilic Organic Carbonates and Thiocarbonates as Vehicles to Convert Hydrosulfide into COS and CS2.

Hydrogen sulfide (H2S) and other reactive sulfur species are important small molecules with biological significance. In addition to common reactive sulfur species like H2S, polysulfides, and persulfides, both carbonyl sulfide (COS) and carbon disulfide (CS2) have been postulated to be potential sources of reduced sulfur. To better understand this possible connection, we demonstrate that H2S can be converted to COS and CS2 by reaction with simple organic carbonate and thiocarbonate electrophiles, respectively.

An Expanded Substrate Spectrum of Sulfide:Quinone Oxidoreductase Found in Pollutant-Degrading Bacteria.

Sulfide:quinone oxidoreductase (Sqr) catalyzes the initial procedure on sulfide transformation, alongside sulfide (H2S, S2-) oxidization coupled with coenzyme Q (CoQ) reducing and reactive sulfur species (RSS) production. Here, we assessed the reactivity of propanethiol (PT) as an alternative substrate for Sqr to maintain intracellular homeostasis in strain S-1 capable of degrading emerging sulfur-containing pollutants. We deleted a gene encoding Sqr, and serial transcriptional difference induced by RSS dynamics was therefore revealed. Next, the reaction properties of two Sqr homologs from strains JMP134 and S-1 were comparatively characterized, respectively. As a result, an additional role of Sqr in yielding RSS from PT was found in reaction mixture prepared by cell-free extracts or purified enzymes. Interestingly, the transformation velocity of PT by Sqr was slower than that of sulfides. From this scenario, it was a rate-determining step that PT as a nucleophilic compound can be added into Sqr cysteine to form disulfide bond and likely serve nonoptimal sulfur recipient. In addition, the role of persulfidation driven by RSS in combating oxidative and sulfur stresses required to be further clarified. Nevertheless, this promiscuity of Sqr-binding organosulfur compounds and its catalytic modulation underscored that expanded substrates might benefit sulfide homeostasis in thiol-degrading bacteria.

A colorimetric fluorescent probe for reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO− and its application in food and bioimaging

In view of the significant role of reactive sulfur species (RSS) and reactive oxygen species (ROS) in maintaining the redox homeostasis of organisms, we proposed a colorimetric fluorescent probe (HTN) for reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO−. C = C is the active site for the Michael addition of HSO3− and the oxidation of ClO−. When HTN interacts with HSO3− and ClO−, it exhibits fluorescence quenching. The addition of oxidizing H2O2 to the system can restore the conjugate structure of the addition product of HSO3− (HTN-HSO3−) and the fluorescence recovery, but it cannot restore the structure of the oxidation product of ClO− (HTN-ClO−). By studying the change of the reversibility/non-reversibility of the probe structure with the addition of H2O2, the purpose of reversible detection of HSO3−/H2O2 and distinguishing HSO3−/ClO− is achieved. In addition, HTN can not only be used as a fluorescent ink to detect HSO3− on the test paper, but also has excellent detection effect on HSO3− and ClO− in real food samples and water samples. Meantime, HTN has good biocompatibility and can target mitochondria to achieve reversible detection of HSO3−/H2O2 and effective discrimination of HSO3−/ClO− in living cells. Therefore, HTN has great potential as a molecular tool for studying redox homeostasis in the interaction network of complex living systems.

Multi-defense Pathways against Electrophiles through Adduct Formation by Low Molecular Weight Substances with Sulfur Atoms.

There is a variety of electrophiles in the environment. In addition, there are precursor chemicals that undergo metabolic activation by enzymes and conversion to electrophiles in the body. Although electrophiles covalently bind to protein nucleophiles, they also form adducts associated with adaptive or toxic responses. Low molecular weight compounds containing sulfur are capable of blocking such adduct formation by capturing the electrophiles. In this review, we present out findings on the capture and inactivation of electrophiles by 1) intracellular glutathione, 2) reactive sulfur species and 3) extracellular cysteine (formed during the production of sulfur adducts). These actions not only substantially suppress electrophilic activity but also regulate protein adduct formation.

Increased hydrogen sulfide turnover serves a cytoprotective role during the development of replicative senescence

The mammalian gasotransmitter hydrogen sulfide (H2S) is produced by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST). Prior studies suggest that H2S may have cytoprotective and anti-aging effects. This project explores the regulation and role of endogenous H2S in a murine model of replicative senescence. H2S and polysulfide levels in RAW 264.7 murine macrophages (control cells: passage 5–10; senescent cells: passage 30–40) were measured using fluorescent probes. The expression of H2S-related enzymes and the activity of senescence marker beta-galactosidase (SA-β-Gal) were also analyzed. CBS, CSE, and 3-MST were inhibited using selective pharmacological inhibitors. Senescence led to a moderate upregulation of CBS and in a significant increase in CSE and 3-MST. H2S degradation enzymes were also elevated in senescence. Inhibition of H2S-producing enzymes reduced H2S levels but increased polysulfides. Inhibition of H2S production during senescence suppressed cell proliferation, and elevated SA-β-Gal and p21 levels. Comparing young and old mice spleens revealed downregulation of CBS and ETHE1 and upregulation of rhodanese and SUOX in older mice. The results demonstrate that increased reactive sulfur turnover occurs in senescent macrophages and that reactive sulfur species supports cell proliferation and regulate cellular senescence

Post-Translational Modifications to Cysteine Residues in Plant Proteins and Their Impact on the Regulation of Metabolism and Signal Transduction.

Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways.

An Update on Glutathione's Biosynthesis, Metabolism, Functions, and Medicinal Purposes.

Glutathione (GSH) has been the focus of increased scientific interest in the last decades. It plays a crucial role in all major physiological processes by supplying antioxidant defenses through participating in cellular redox reactions in the human body and other living organisms. GSH also participates in detoxifying xenobiotics, protecting protein thiols from crosslinking and oxidation, regulating the cell cycle, storing cysteine, etc. The significant role of GSH in the most important physiological processes has been highlighted, such as maintaining the redox balance and reducing oxidative stress due to its ability to inactivate the reactive oxygen, nitrogen, and sulfur species. It can also enhance metabolic detoxification and regulate the function of the immune system. All of these characteristics make it a universal biomarker since its proper balance is essential for improving health and treating some age-related disorders. This review presents a current concept of the synthesis and metabolism of GSH; its main functions in a living organism, and as a precursor and cofactor; data on the use of GSH for medicinal purposes in the prevention and treatment of some diseases, as well as a nutritional strategy to maintain a normal pool of GSH in the body. The data were gathered by searching relevant information in multiple databases, such as PubMed, Scopus, ScienceDirect, and Google Scholar.

A near-infrared ratiometric fluorescent probe for the sensing and imaging of sulfur dioxide derivatives in living systems

As a reactive sulfur species, sulfur dioxide (SO2) and its derivatives play crucial role in various physiological processes, which can maintain redox homeostasis at normal levels and lead to the occurrence of many diseases at abnormal levels. So, the development of a suitable fluorescent probe is a crucial step in advancing our understanding of the role of SO2 derivatives in living organisms. Herein, we developed a near-infrared fluorescent probe (SP) based on the ICT mechanism to monitor SO2 derivatives in living organisms in a ratiometric manner. The probe SP exhibited excellent selectivity, good sensitivity, fast response rate (within 50 s), and low detection limit (1.79 µM). In addition, the cell experiment results suggested that the SP has been successfully employed for the real-time monitoring of endogenous and exogenous SO2 derivatives with negligible cytotoxicity. Moreover, SP was effective in detecting SO2 derivatives in mice.

The SOD1 Inhibitor, LCS-1, Oxidizes H2S to Reactive Sulfur Species, Directly and Indirectly, through Conversion of SOD1 to an Oxidase.

LCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H2S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H2S and SOD1 using thiol-specific fluorophores, liquid chromatography-mass spectrometry, electron paramagnetic resonance (EPR), UV-vis spectrometry, and oxygen consumption. We show that LCS-1 catalytically oxidizes H2S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H2Sn, n = 2-6). These reactions consume oxygen and produce hydrogen peroxide, but they do not have an EPR signature, nor do they affect the UV-vis spectrum. Surprisingly, LCS-1 synergizes with SOD1, but not SOD2, to oxidize H2S to H2S3-6. LCS-1 forms monothiol adducts with H2S, glutathione (GSH), and cysteine (Cys), but not with oxidized glutathione or cystine; both thiol adducts inhibit LCS-1-SOD1 synergism. We propose that LCS-1 forms an adduct with SOD1 that disrupts the intramolecular Cys57-Cys146 disulfide bond and transforms SOD1 from a dismutase to an oxidase. This would increase cellular ROS and polysulfides, the latter potentially affecting cellular signaling and/or cytoprotection.

High-Altitude Medicinal Plants as Promising Source of Phytochemical Antioxidants to Combat Lifestyle-Associated Oxidative Stress-Induced Disorders.

Oxidative stress, driven by reactive oxygen, nitrogen, and sulphur species (ROS, RNS, RSS), poses a significant threat to cellular integrity and human health. Generated during mitochondrial respiration, inflammation, UV exposure and pollution, these species damage cells and contribute to pathologies like cardiovascular issues, neurodegeneration, cancer, and metabolic syndromes. Lifestyle factors exert a substantial influence on oxidative stress levels, with mitochondria emerging as pivotal players in ROS generation and cellular equilibrium. Phytochemicals, abundant in plants, such as carotenoids, ascorbic acid, tocopherols and polyphenols, offer diverse antioxidant mechanisms. They scavenge free radicals, chelate metal ions, and modulate cellular signalling pathways to mitigate oxidative damage. Furthermore, plants thriving in high-altitude regions are adapted to extreme conditions, and synthesize secondary metabolites, like flavonoids and phenolic compounds in bulk quantities, which act to form a robust antioxidant defence against oxidative stress, including UV radiation and temperature fluctuations. These plants are promising sources for drug development, offering innovative strategies by which to manage oxidative stress-related ailments and enhance human health. Understanding and harnessing the antioxidant potential of phytochemicals from high-altitude plants represent crucial steps in combating oxidative stress-induced disorders and promoting overall wellbeing. This study offers a comprehensive summary of the production and physio-pathological aspects of lifestyle-induced oxidative stress disorders and explores the potential of phytochemicals as promising antioxidants. Additionally, it presents an appraisal of high-altitude medicinal plants as significant sources of antioxidants, highlighting their potential for drug development and the creation of innovative antioxidant therapeutic approaches.

Oxidative Cysteine Post Translational Modifications Drive the Redox Code Underlying Neurodegeneration and Amyotrophic Lateral Sclerosis.

Redox dysregulation, an imbalance between oxidants and antioxidants, is crucial in the pathogenesis of various neurodegenerative diseases. Within this context, the "redoxome" encompasses the network of redox molecules collaborating to maintain cellular redox balance and signaling. Among these, cysteine-sensitive proteins are fundamental for this homeostasis. Due to their reactive thiol groups, cysteine (Cys) residues are particularly susceptible to oxidative post-translational modifications (PTMs) induced by free radicals (reactive oxygen, nitrogen, and sulfur species) which profoundly affect protein functions. Cys-PTMs, forming what is referred to as "cysteinet" in the redox proteome, are essential for redox signaling in both physiological and pathological conditions, including neurodegeneration. Such modifications significantly influence protein misfolding and aggregation, key hallmarks of neurodegenerative diseases such as Alzheimer's, Parkinson's, and notably, amyotrophic lateral sclerosis (ALS). This review aims to explore the complex landscape of cysteine PTMs in the cellular redox environment, elucidating their impact on neurodegeneration at protein level. By investigating specific cysteine-sensitive proteins and the regulatory networks involved, particular emphasis is placed on the link between redox dysregulation and ALS, highlighting this pathology as a prime example of a neurodegenerative disease wherein such redox dysregulation is a distinct hallmark.

Rational design of cascade reaction-assisted trinal-site fluorescent probe for simultaneous discrimination of Cys, Hcy, GSH, and H2S in living cells and zebrafish

The reactive sulfur species (RSS), in particular Cys, Hcy, GSH, and H2S, play an essential role in lots of critical physiological and pathological processes. To elucidate their complex interrelationships in signaling and metabolic pathways, suitable molecular tools to simultaneously distinguish Cys, Hcy, GSH, and H2S are urgently needed. Herein, we developed a novel cascade reaction-assisted trinal-site fluorescent probe BCS by connecting three functional fragments for simultaneously distinguishing Cys, Hcy, GSH, and H2S. The probe BCS exhibited red fluorescence in the absence of the four biological RSS. However, with the attendance of Cys, Hcy, GSH, and H2S, the probe showed different fluorescence patterns, including blue, blue/red, green/red, and no fluorescence, respectively. More importantly, based on the different RSS-induced fluorescence patterns and multi-color fluorescence imaging, probe BCS was used to discriminate and visualize all four biological RSS in both HepG2 cells and zebrafish. It is expected that probe BCS will have applications in the diagnosis and research of RSS-related diseases in the future.

α-Lipoic acid increases the viability of nephrocytes and elevates sulfane sulfur level in plasma of patients with chronic kidney disease.

Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.

Transpersulfidation or H2S Release? Understanding the Landscape of Persulfide Chemical Biology.

Persulfides (RSSH) are biologically important reactive sulfur species that are endogenously produced, protect key cysteine residues from irreversible oxidation, and are important intermediates during different enzymatic processes. Although persulfides are stronger nucleophiles than their thiol counterparts, persulfides can also act as electrophiles in their neutral, protonated form in specific environments. Moreover, persulfides are electrophilic at both sulfur atoms, and the reaction with a thiolate can lead to either H2S release with disulfide formation or alternatively result in transpersulfidation. Despite the broad acceptance of these reaction pathways, the specific properties that control whether persulfides react through the H2S-releasing or transpersulfidation pathway remain elusive. Herein, we use a combined computational and experimental approach to directly investigate the reactivity between persulfides and thiols to answer these questions. Using density functional theory (DFT) calculations, we demonstrate that increasing steric bulk or electron withdrawal near the persulfide can shunt persulfide reactivity through the transpersulfidation pathway. Building from these insights, we use a synthetic persulfide donor and an N-iodoacetyl l-tyrosine methyl ester (TME-IAM) trapping agent to experimentally monitor and measure transpersulfidation from a bulky penicillamine-based persulfide to a cysteine-based thiol, which, to the best of our knowledge, is the first direct observation of transpersulfidation between low-molecular-weight species. Taken together, these combined approaches highlight how the properties of persulfides are directly impacted by local environments, which has significant impacts in understanding the complex chemical biology of these reactive species.

Metagenome mining and functional analysis reveal oxidized guanine DNA repair at the Lost City Hydrothermal Field.

The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H2 and low levels of O2 in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome-assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O2 levels rose following the great oxidation event.

A ratiometric fluorescent probe with dual-targeting capability for heat shock imaging

HSO3− is an important reactive sulfur species that maintains the normal physiological activities of living organisms and participates in a variety of redox homeostatic processes. It has been found that changes in HSO3− levels is closely related to the heat stroke phenomenon of the organism. Heat stroke causes damage to normal cells, which in turn causes damage to the body and even death. It is crucial to accurately monitor and track the physiological behavior of HSO3− during heat stroke. Herein, a ratiometric multifunctional fluorescent probe DRM-SO2 with dual-targeting ability to rapidly and precisely recognize HSO3− being constructed based on the FRET mechanism. DRM-SO2 has extra Large Stokes shift (216 nm), very high sensitivity (DL = 12.2 nM), fast response time and good specificity. When DRM-SO2 undergoes Michael addition with HSO3−, the fluorescence emission peak was blue-shifted from 616 nm to 472 nm, and a clear ratiometric signal appeared. The interaction between lysosomes and mitochondria in maintaining cellular homeostasis was investigated by the dual-targeting ability of the probe using HSO3− as a mediator. DRM-SO2 achieved successful targeting and real-time monitoring of exogenous and endogenous HSO3− in the cells. More importantly, imaging experiments in heat stroke mice revealed high HSO3− expression in intestinal tissues. This provides new ideas and research tools for early prevention of heat stroke-induced diseases such as intestinal injuries. In addition, the semi-quantitative monitoring experiments for paper-based visualization of HSO3− make the probe promising for the design of portable detectors.

An Expanded Palette of Fluorescent COS/H2S‐Releasing Donors for H2S Delivery, Detection, and In Vivo Application

AbstractHydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol‐activated fluorescence turn‐on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared‐emitting dyes functionalized with an H2S‐releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn‐on response (3–310‐fold) and high H2S release efficiencies (>60 %). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non‐transparent organisms.

An Expanded Palette of Fluorescent COS/H2S-Releasing Donors for H2S Delivery, Detection, and In Vivo Application.

Hydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol-activated fluorescence turn-on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared-emitting dyes functionalized with an H2S-releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn-on response (3-310-fold) and high H2S release efficiencies (>60 %). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non-transparent organisms.