Sodium thiosulfate: A donor or carrier signaling molecule for hydrogen sulfide?

Sodium thiosulfate has been used for decades in the treatment of calciphylaxis and cyanide detoxification, and has recently shown initial therapeutic promise in critical diseases such as neuronal ischemia, diabetes mellitus, heart failure and acute lung injury. However, the precise mechanism of sodium thiosulfate remains incompletely defined and sometimes contradictory. Although sodium thiosulfate has been widely accepted as a donor of hydrogen sulfide (H2S), emerging findings suggest that it is the executive signaling molecule for H2S and that its effects may not be dependent on H2S. This article presents an overview of the current understanding of sodium thiosulfate, including its synthesis, biological characteristics, and clinical applications of sodium thiosulfate, as well as the underlying mechanisms in vivo. We also discussed the interplay of sodium thiosulfate and H2S. Our review highlights sodium thiosulfate as a key player in sulfide signaling with the broad clinical potential for the future.

Reaction Mechanisms of H2S Oxidation by Naphthoquinones.

1,4-naphthoquinones (NQs) catalytically oxidize H2S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H2S oxidation by NQs with various substituent groups. In general, the order of H2S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H2S to polysulfides and sulfoxides. This suggests that H2S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H2S2-4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H2S impurities in the polysulfides or an equilibrium between H2S and H2Sn. Collectively, NQ oxidation of H2S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S8 colloid is formed.

Recent progress and outlooks in rhodamine-based fluorescent probes for detection and imaging of reactive oxygen, nitrogen, and sulfur species.

Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) serve as vital mediators essential for preserving intracellular redox homeostasis within the human body, thereby possessing significant implications across physiological and pathological domains. Nevertheless, deviations from normal levels of ROS, RNS, and RSS disturb redox homeostasis, leading to detrimental consequences that compromise bodily integrity. This disruption is closely linked to the onset of various human diseases, thereby posing a substantial threat to human health and survival. Small-molecule fluorescent probes exhibit considerable potential as analytical instruments for the monitoring of ROS, RNS, and RSS due to their exceptional sensitivity and selectivity, operational simplicity, non-invasiveness, localization capabilities, and ability to facilitate in situ optical signal generation for real-time dynamic analyte monitoring. Due to their distinctive transition from their spirocyclic form (non-fluorescent) to their ring-opened form (fluorescent), along with their exceptional light stability, broad wavelength range, high fluorescence quantum yield, and high extinction coefficient, rhodamine fluorophores have been extensively employed in the development of fluorescent probes. This review primarily concentrates on the investigation of fluorescent probes utilizing rhodamine dyes for ROS, RNS, and RSS detection from the perspective of different response groups since 2016. The scope of this review encompasses the design of probe structures, elucidation of response mechanisms, and exploration of biological applications.

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.

In pursuit of feedback activation: New insights into redox-responsive hydropersulfide prodrug combating oxidative stress.

Redox-responsive hydropersulfide prodrugs are designed to enable a more controllable and efficient hydropersulfide (RSSH) supply and to thoroughly explore their biological and therapeutic applications in oxidative damage. To obtain novel activation patterns triggered by redox signaling, we focused on NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1), a canonical antioxidant enzyme, and designed NQO1-activated RSSH prodrugs. We also performed a head-to-head comparison of two mainstream structural scaffolds with solid quantitative analysis of prodrugs, RSSH, and metabolic by-products by LC-MS/MS, confirming that the perthiocarbamate scaffold was more effective in intracellular prodrug uptake and RSSH production. The prodrug was highly potent in oxidative stress management against cisplatin-induced nephrotoxicity. Strikingly, this prodrug possessed potential feedback activation properties by which the delivered RSSH can further escalate the prodrug activation via NQO1 upregulation. Our strategy pushed RSSH prodrugs one step further in the pursuit of efficient release in biological matrices and improved druggability against oxidative stress.

Effect of reactive oxygen, nitrogen, and sulfur species on signaling pathways in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease in which fats, lipids, cholesterol, calcium, proliferating smooth muscle cells, and immune cells accumulate in the intima of the large arteries, forming atherosclerotic plaques. A complex interplay of various vascular and immune cells takes place during the initiation and progression of atherosclerosis. Multiple reports indicate that tight control of reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) production is critical for maintaining vascular health. Unrestricted ROS and RNS generation may lead to activation of various inflammatory signaling pathways, facilitating atherosclerosis. Given these deleterious consequences, it is important to understand how ROS and RNS affect the signaling processes involved in atherogenesis. Conversely, RSS appears to exhibit an atheroprotective potential and can alleviate the deleterious effects of ROS and RNS. Herein, we review the literature describing the effects of ROS, RNS, and RSS on vascular smooth muscle cells, endothelial cells, and macrophages and focus on how changes in their production affect the initiation and progression of atherosclerosis. This review also discusses the contribution of ROS, RNS, and RSS in mediating various post-translational modifications, such as oxidation, nitrosylation, and sulfation, of the molecules involved in inflammatory signaling.

Impact of Reactive Sulfur Species on Entamoeba histolytica: Modulating Viability, Motility, and Biofilm Degradation Capacity.

Reactive sulfur species (RSS) like hydrogen sulfide (H2S) and cysteine persulfide (Cys-SSH) emerged as key signaling molecules with diverse physiological roles in the body, depending on their concentration and the cellular environment. While it is known that H2S and Cys-SSH are produced by both colonocytes and by the gut microbiota through sulfur metabolism, it remains unknown how these RSS affect amebiasis caused by Entamoeba histolytica, a parasitic protozoan that can be present in the human gastrointestinal tract. This study investigates H2S and Cys-SSH's impact on E. histolytica physiology and explores potential therapeutic implications. Exposing trophozoites to the H2S donor, sodium sulfide (Na2S), or to Cys-SSH led to rapid cytotoxicity. A proteomic analysis of Cys-SSH-challenged trophozoites resulted in the identification of >500 S-sulfurated proteins, which are involved in diverse cellular processes. Functional assessments revealed inhibited protein synthesis, altered cytoskeletal dynamics, and reduced motility in trophozoites treated with Cys-SSH. Notably, cysteine proteases (CPs) were significantly inhibited by S-sulfuration, affecting their bacterial biofilm degradation capacity. Immunofluorescence microscopy confirmed alterations in actin dynamics, corroborating the proteomic findings. Thus, our study reveals how RSS perturbs critical cellular functions in E. histolytica, potentially influencing its pathogenicity and interactions within the gut microbiota. Understanding these molecular mechanisms offers novel insights into amebiasis pathogenesis and unveils potential therapeutic avenues targeting RSS-mediated modifications in parasitic infections.

Analysis and characterization of sulfane sulfur.

In the late 1970s, sulfane sulfur was defined as sulfur atoms covalently bound only to sulfur atoms. However, this definition was not generally accepted, as it was slightly vague and difficult to comprehend. Thus, in the early 1990s, it was defined as "bound sulfur," which easily converts to hydrogen sulfide upon reduction with a thiol-reducing agent. H2S-related bound sulfur species include persulfides (R-SSH), polysulfides (H2Sn, n ≥ 2 or R-S(S)nS-R, n ≥ 1), and protein-bound elemental sulfur (S0). Many of the biological effects currently associated with H2S may be attributed to persulfides and polysulfides. In the 20th century, quantitative determination of "sulfane sulfur" was conventionally performed using a reaction called cyanolysis. Several methods have been developed over the past 30 years. Current methods used for the detection of H2S and polysulfides include colorimetric assays for methylene blue formation, sulfide ion-selective or polarographic electrodes, gas chromatography with flame photometric or sulfur chemiluminescence detection, high-performance liquid chromatography analysis with fluorescent derivatization of sulfides, liquid chromatography with tandem mass spectrometry, the biotin switch technique, and the use of sulfide or polysulfide-sensitive fluorescent probes. In this review, we discuss the methods reported to date for measuring sulfane sulfur and the results obtained using these methods.

Synergistic Activation of Nitrite and Thiocarbonyl Compounds Affords NO and Sulfane Sulfur via (Per)thionitrite (SNO- /SSNO- ).

(Per)thionitrite (SNO- /SSNO- ) intermediates play vital roles in modulating nitric oxide (NO) and hydrogen sulfide (H2 S) dependent bio-signalling processes. Whilst the previous preparations of such intermediates involved reactive H2 S/HS- or sulfane sulfur (S0 ) species, the present report reveals that relatively stable thiocarbonyl compounds (such as carbon disulfide (CS2 ), thiocarbamate, thioacetic acid, and thioacetate) react with nitrite anion to yield SNO- /SSNO- . For instance, the reaction of CS2 and nitrite anion (NO2 - ) under ambient condition affords CO2 and SNO- /SSNO- . A detailed investigation involving UV/Vis, FTIR, HRMS, and multinuclear NMR studies confirm the formation of SNO- /SSNO- , which are proposed to form through an initial nucleophilic attack by nitrite anion followed by a transnitrosation step. Notably, reactions of CS2 and nitrite in the presence of thiol RSH show the formation of organic polysulfides R-Sn -R, thereby illustrating that the thiocarbonyls are capable of influencing the pool of bioavailable sulfane sulfurs. Furthermore, the availability of both NO2 - and thiocarbonyl motifs in the biological context hints at their synergistic metal-free activations leading to the generation of NO gas and various reactive sulfur species via SNO- /SSNO- .

Reactive Sulfur Species Produced by Cystathionine γ-lyase Function in the Establishment of Mesorhizobium loti-Lotus japonicus Symbiosis.

Reactive sulfur species (RSS) are present in root nodules; however, their role in symbiosis and the mechanisms underlying their production remain unclear. We herein investigated whether RSS produced by the cystathionine γ-lyase (CSE) of microsymbionts are involved in root nodule symbiosis. A cse mutant of Mesorhizobium loti exhibited the decreased production of hydrogen sulfide and other RSS. Although the CSE mutation of M. loti did not affect the early stages of symbiosis, i.e., infection and nodulation, with Lotus japonicus, it reduced the nitrogenase activity of nodules and induced their early senescence. Additionally, changes in the production of sulfur compounds and an increase in reactive oxygen species (ROS) were observed in the infected cells of nodules induced by the cse mutants. The effects of CSE inhibitors in the L. japonicus rhizosphere on symbiosis with M. loti were also investigated. All three CSE inhibitors suppressed infection and nodulation by M. loti concomitant with decreased RSS levels and increased ROS and nitric oxide levels. Therefore, RSS derived from the CSE activity of both the microsymbiont and host plant are required for symbiosis, but function at different stages of symbiosis, possibly with crosstalk with other reactive mole-cular species.

In the Alphaproteobacterium Hyphomicrobium denitrificans SoxR Serves a Sulfane Sulfur-Responsive Repressor of Sulfur Oxidation.

In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of genes for enzymes involved in sulfur oxidation is needed to adjust metabolic flux to environmental conditions. However, little is known about the sensing and response to inorganic sulfur compounds such as thiosulfate in sulfur-oxidizing bacteria. In the Alphaproteobacterium Hyphomicrobium denitrificans, one strategy is the use of the ArsR-SmtB-type transcriptional regulator SoxR. We show that this homodimeric repressor senses sulfane sulfur and that it is crucial for the expression not only of sox genes encoding the components of a truncated periplasmic thiosulfate-oxidizing enzyme system but also of several other sets of genes for enzymes of sulfur oxidation. DNA binding and transcriptional regulatory activity of SoxR are controlled by polysulfide-dependent cysteine modification. The repressor uses the formation of a sulfur bridge between two conserved cysteines as a trigger to bind and release DNA and can also form a vicinal disulfide bond to orchestrate a response to oxidizing conditions. The importance of the sulfur bridge forming cysteines was confirmed by site-directed mutagenesis, mass spectrometry, and gel shift assays. In vivo, SoxR interacts directly or indirectly with a second closely related repressor, sHdrR.

Comparison of Reactive Sites in 2(1H)-Quinolone Derivatives for the Detection of Biologically Important Sulfur Compounds.

Novel fluorescent probes based on 2(1H)-quinolone skeleton containing a malonate group (Q1-Q3) were synthesized and proposed for biothiols detection. Their chemical reactivity toward thiols was compared to the reactivity of derivative having a dicyanovinyl group (Q4) as a reactive site. The detailed photophysical properties of these compounds were assessed through the determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. In the presence of biothiols, an increase in the fluorescence intensity of compounds Q1-Q3 and a hypsochromic shift in their emission bands were observed. In contrast, the compound with the dicyanovinyl group (Q4) in the presence of biothiols and cyanide ion showed the quenching of fluorescence, while a fluorescence "turn on" effect was observed toward reactive sulfur species.

Treatment with aripiprazole and N-acetylcysteine affects anaerobic cysteine metabolism in the hippocampus and reverses schizophrenia-like behavior in the neurodevelopmental rat model of schizophrenia.

Preclinical and clinical studies have shown that the antipsychotic drug aripiprazole and the antioxidant N-acetylcysteine have unique biological properties. The aim of the study was to investigate, in a rat model of schizophrenia, the effects of chronic administration of these drugs on schizophrenia-like behaviors and anaerobic cysteine metabolism in the hippocampus (HIP). The schizophrenia-type changes were induced in Sprague-Dawley rats by repeated administration of the glutathione synthesis inhibitor l-butionine-(S,R)-sulfoximine in combination with the dopamine reuptake inhibitor GBR 12909 in the early postnatal period. Adult model rats were chronically treated with aripiprazole (0.3 mg·kg-1 , i.p.) or N-acetylcysteine (30 mg·kg-1 , orally), and their effects on schizophrenia-like behaviors were assessed using the social interaction test and novel object recognition test. In the HIP, the level of anaerobic cysteine metabolites, H2 S, and bound sulfane sulfur were determined by a fluorescence method, while the expression of H2 S-synthetizing enzymes: cystathionine ß-synthase (CBS) and mercaptopyruvate sulfurtransferase (MST) by western blot. Long-term treatment with aripiprazole or N-acetylcysteine reversed social and cognitive deficits and reduced the exploratory behaviors. In the HIP of 16-day-old model pups, H2 S levels and MST protein expression were significantly decreased. In adult model rats, H2 S levels remained unchanged, bound sulfane sulfur significantly increased, and the expression of CBS and MST slightly decreased. The studied drugs significantly reduced the level of bound sulfane sulfur and the expression of tested enzymes. The reduction in bound sulfane sulfur level coincided with the attenuation of exploratory behavior, suggesting that modulation of anaerobic cysteine metabolism in the HIP may have therapeutic potential in schizophrenia.

Reactive Sulfur Species in Human Diseases.

Significance: Reactive sulfur species (RSS) have been recently recognized as redox molecules no less important than reactive oxygen species or reactive nitrogen species. They possess regulatory and protective properties and are involved in various metabolic processes, thereby contributing to the maintenance of human health. It has been documented that many disorders, including neurological, cardiovascular, and respiratory diseases, diabetes mellitus (DM), and cancer, are related to the disruption of RSS homeostasis. Recent Advances: There is still a growing interest in the role of RSS in human diseases. Since a decrease in hydrogen sulfide or other RSS has been reported in many disorders, safe and efficient RSS donors have been developed and tested under in vitro conditions or on animal models. Critical Issues: Cardiovascular diseases and DM are currently the most common chronic diseases worldwide due to stressful and unhealthy lifestyles. In addition, because of high prevalence and aging of the population, neurological disorders including Parkinson's disease and Alzheimer's disease as well as respiratory diseases are a formidable challenge for health care systems. From this point of view, the knowledge of the role of RSS in these disorders and RSS modulation options are important and could be useful in therapeutic strategies. Future Directions: Improvement and standardization of analytical methods used for RSS estimation are crucial for the use of RSS as diagnostic biomarkers. Finding good, safe RSS donors applicable for therapeutic purposes could be useful as primary or adjunctive therapy in many common diseases. Antioxid. Redox Signal. 39, 1000-1023.

Sensing and regulation of reactive sulfur species (RSS) in bacteria.

The infected host deploys generalized oxidative stress caused by small inorganic reactive molecules as antibacterial weapons. An emerging consensus is that hydrogen sulfide (H2S) and forms of sulfur with sulfur-sulfur bonds termed reactive sulfur species (RSS) provide protection against oxidative stressors and antibiotics, as antioxidants. Here, we review our current understanding of RSS chemistry and its impact on bacterial physiology. We start by describing the basic chemistry of these reactive species and the experimental approaches developed to detect them in cells. We highlight the role of thiol persulfides in H2S-signaling and discuss three structural classes of ubiquitous RSS sensors that tightly regulate cellular H2S/RSS levels in bacteria, with a specific focus on the chemical specificity of these sensors.

Emergence of (hydro)persulfides as suppressors of lipid peroxidation and ferroptotic cell death.

Recognition of the prevalence of hydropersulfides (RSSH) and characterization of their enhanced two-electron reactivity relative to thiols have led to their implication in maintaining cellular redox homeostasis, in addition to other potential roles. Recent attention on the one-electron reactivity of RSSH has uncovered their potent radical-trapping antioxidant activity, which enables them to inhibit phospholipid peroxidation and associated cell death by ferroptosis. Herein, we briefly review key aspects of the reactivity and underlying physicochemical properties of RSSH. We emphasize their reactivity to radicals-particularly lipid peroxyl radicals that propagate the lipid peroxidation chain reaction-and the recent recognition that this results in ferroptosis suppression. We highlight open questions related to recent developments in this area and, given that all living organisms possess the ability to synthesize persulfides endogenously, suggest they may be primordial radical scavengers that occurred early in evolution and still play a role today.

Sulfur-Containing Persistent Free Radicals and Reactive Species on Photoaged Microplastics: Identification and the Formation Mechanism.

The elemental composition may affect the persistent free radical (PFR) and reactive species (RS) formation associated with photoaging microplastics; however, a relevant study is still lacking. This study systematically investigated the formation, evolution, and types of PFRs and RS on sulfur-containing microplastics (S-MPs) under simulated sunlight. Electron paramagnetic resonance detection and power saturation curve analysis isolated three different PFRs on each photoaging poly(phenylene sulfide) (PPS) and polysulfone (PSF). Combining the results of characterization and density functional theory calculation, these observed PFRs on the irradiated S-MPs were classified as oxygen-centered radicals with an adjacent S atom (namely, thio-oxygen radicals), oxygen-centered and sulfur-centered radicals, where the thio-oxygen radicals on PPS were benzenethiol-like radicals, and oxygen-centered radicals and sulfur-centered radicals on PSF that were identified as benzenesulfonic-like radicals and phenyl sulfonyl-like radicals, respectively. Moreover, potential precursor molecule fragments of PFRs on the photoaging S-MPs, including p-toluenesulfinic acid and benzenesulfonic acid, were detected by pyrolysis-gas chromatography/mass spectrometry and liquid chromatography-mass spectrometry. Interestingly, reactive sulfur species (SO3•-) was also observed on irradiated S-MPs in addition to reactive oxygen species, which was mainly derived from the reaction of •OH and sulfonyl radicals. These results have implications for assessing the potential risks of atmospheric S-MPs.

Diverse Possibilities of Si-Based Agent, a Unique New Antioxidant.

Antioxidant therapy is an effective approach for treating diseases in which oxidative stress is involved in the onset of symptoms. This approach aims to rapidly replenish the antioxidant substances in the body when they are depleted due to excess oxidative stress. Importantly, a supplemented antioxidant must specifically eliminate harmful reactive oxygen species (ROS) without reacting with physiologically beneficial ROS, which are important to the body. In this regard, typically used antioxidant therapies can be effective, but may cause adverse effects due to their lack of specificity. We believe that Si-based agents are epoch-making drugs that can overcome these problems associated with current antioxidative therapy. These agents alleviate the symptoms of oxidative-stress-associated diseases by generating large amounts of the antioxidant hydrogen in the body. Moreover, Si-based agents are expected to be highly effective therapeutic drug candidates because they have anti-inflammatory, anti-apoptotic, and antioxidant effects. In this review, we discuss Si-based agents and their potential future applications in antioxidant therapy. There have been several reports of hydrogen generation from silicon nanoparticles, but unfortunately, none have been approved as pharmaceutical agents. Therefore, we believe that our research into medical applications using Si-based agents is a breakthrough in this research field. The knowledge obtained thus far from animal models of pathology may greatly contribute to the improvement of existing treatment methods and the development of new treatment methods. We hope that this review will further revitalize the research field of antioxidants and lead to the commercialization of Si-based agents.

Reactive Sulfur Species Omics Analysis in the Brain Tissue of the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder whereby oxidative stress augmentation results in mitochondrial dysfunction and cell death by apoptosis. Emerging evidence indicates that reactive sulfur species (RSS), such as glutathione hydropersulfide (GSSH), is endogenously produced, functions as potent antioxidants, and regulate redox signaling through the formation of protein polysulfides. However, the relationship between RSS and AD pathogenesis is not fully understood. In this study, we analyzed endogenous RSS production in the brain tissue of a familial AD model (5xFAD) mouse using multiple RSS-omics approaches. Memory impairment, increased amyloid plaques, and neuroinflammation have been confirmed in 5xFAD mice. Quantitative RSS omics analysis revealed that the total polysulfide content was significantly decreased in the brains of 5xFAD mice, whereas there was no significant difference in the levels of glutathione, GSSH, or hydrogen sulfide between wild-type and 5xFAD mice. In contrast, a significant decline in the protein polysulfide status was observed in the brains of 5xFAD mice, suggesting that RSS production and subsequent redox signaling might be altered during the onset and progression of AD. Our findings have important implications for understanding the significance of RSS in the development of preventive and therapeutic strategies for AD.

Reduction of metmyoglobin by inorganic disulfide species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.