Reactive Thiol Research Articles

Simultaneous Quantitation of Multiple Biological Thiols Using Reactive Ionization and Derivatization with Charged Mass Tags.

The biologically important thiols (cysteine, homocysteine, N-acetyl cysteine, and glutathione) are key species in redox homeostasis, and there is a clinical need to measure them rapidly, accurately, and simultaneously at low levels in complex biofluids. The solution to the challenge presented here is based on a new derivatizing reagent that combines a thiol-selective unit to optimize the chemical transformation and a precharged pyridinium unit chosen to maximize sensitivity in mass spectrometry. Derivatization is performed simultaneously with ionization ("reactive ionization"), and mass spectrometry is used to record and characterize the thiol reaction products. The method is applicable over the concentration range from 1 μM to 10 mM and is demonstrated for 25 blood serum, 1 plasma, and 3 types of tissue samples. The experiment is characterized by limited sample preparation (<4 min) and short analysis time (<1 min). High precision and accuracy (both better than 8%) are validated using independent HPLC-MS analysis. Cystine-cysteine redox homeostasis can be monitored by introducing an additional reduction step, and the accuracy and precision of these results are also validated by HPLC-MS.

Effects of aging and repeated freeze-thaw cycles on quality attributes, physicochemical and biochemical properties, and sensory characteristics of beef sirloins

This study aimed to evaluate the influence of aging before freezing and repeated freezing on quality attributes of beef sirloins. At 5 days postmortem, pairs of sirloins (M. gluteus medius) were collected from 20 beef carcasses (USDA Choice grade). The sirloin samples from each side were assigned to either no additional aging (A0) or 4 weeks of aging (A4). Samples were then subjected to either single freeze-thawing (A0FT and A4FT) or repeated freeze-thawing (A0FT × 2 and A4FT × 2) over 3 weeks. Following aging (2 °C), freezing (−20 °C), and thawing (2 °C), the samples were evaluated for water-holding capacity (WHC), Warner-Bratzler shear force (WBSF), myofibrillar fragmentation index (MFI), color, 2-thiobarbituric acid reactive substances (TBARS), carbonyl and thiol contents, and consumer sensory evaluation (n = 120). Freezing without aging increased freeze-thaw and drip losses in A0FT and A0FT × 2 compared to A4FT and A4FT × 2 (p < 0.05). Freezing and repeated freezing decreased WBSF values (p < 0.05) regardless of aging status. Similarly, MFI was higher in A0FT and A4FT compared to A0 and A4, respectively (p < 0.05). Both freezing and repeated freezing increased TBARS levels (p < 0.05). Moreover, freezing without aging reduced CIE L*, a*, b* and chroma values (p < 0.05). However, aged/frozen samples retained similar color attributes to never frozen counterparts (p > 0.05). Consumer sensory panel reported improved sensory attributes of aged/frozen samples, without any adverse effects from repeated freezing. These findings indicate that aging can improve WHC, color, and tenderness of single or repeated frozen/thawed beef sirloins, suggesting it as a practical strategy for the industry to improve meat quality and value of frozen/thawed beef.

Scalable Thiol Reactivity Profiling Identifies Azetidinyl Oxadiazoles as Cysteine-Targeting Electrophiles.

Cysteine reactive groups are a mainstay in the design of covalent drugs and probe molecules, yet only a handful of electrophiles are routinely used to target this amino acid. Here, we report the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. High throughput screening using STRP identified the azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. We show the utility of this reactive group with the discovery of an azetidinyl oxadiazole containing a small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. This study adds a novel cysteine targeting group to the electrophilic lexicon and provides robust methodology to rapidly surveil libraries for reactivity with cysteine.

Interweavable Metalloporphyrin‐based Fibers for Indirect Electrocatalysis

The applications of indirect electrocatalysis toward potential industrial processes are drastically limited by the utilization or processing forms of electrocatalysts. The remaining challenges of electrocatalysts like the recycling in homogeneous systems or pulverization in heterogeneous systems call for advanced processing forms to meet the desired requirements. Here, we report a series of metalloporphyrin‐based polymer fibers (M‐PF, M = Ni, Cu and Zn) through a rigid‐flexible polymerization strategy based on rigid metalloporphyrin and flexible thiourea units that can be applied as heterogenous redox‐mediators in indirect electrocatalysis. These functional fibers with high strength and flexibility exhibit interweavable and designable functions that can be processed into different fiber‐forms like knotted, two‐spiral, three‐ply, five‐ply fibers or even interweaved networks. Interestingly, they can be readily applied in S‐S bond cleaving/cyclization reaction or extended oxidative self‐coupling reaction of thiols with high efficiency. Remarkably, it enables the scale‐up production (1.25 g in a batch‐experiment) under laboratory conditions.

Interweavable Metalloporphyrin-Based Fibers for Indirect Electrocatalysis.

The applications of indirect electrocatalysis toward potential industrial processes are drastically limited by the utilization or processing forms of electrocatalysts. The remaining challenges of electrocatalysts like the recycling in homogeneous systems or pulverization in heterogeneous systems call for advanced processing forms to meet the desired requirements. Here, we report a series of metalloporphyrin-based polymer fibers (M-PF, M=Ni, Cu and Zn) through a rigid-flexible polymerization strategy based on rigid metalloporphyrin and flexible thiourea units that can be applied as heterogeneous redox-mediators in indirect electrocatalysis. These functional fibers with high strength and flexibility exhibit interweavable and designable functions that can be processed into different fiber-forms like knotted, two-spiral, three-ply, five-ply fibers or even interweaved networks. Interestingly, they can be readily applied in S-S bond cleaving/cyclization reaction or extended oxidative self-coupling reaction of thiols with high efficiency. Remarkably, it enables the scale-up production (1.25 g in a batch-experiment) under laboratory conditions.

Identification of novel microcystins in algal extracts by a liquid chromatography–high-resolution mass spectrometry data analysis pipeline

BackgroundMicrocystins are an emergent public health problem. These toxins are secondary metabolites of harmful cyanobacterial blooms, with blooms becoming more prevalent with eutrophication of water. Exposure to microcystins can result in sickness, liver damage, and even death. Over 300 microcystins have been identified to date, with differences in toxicity based on the specific amino acid composition. Because of this diversity in microcystins, as well as the likelihood of detecting as yet undiscovered microcystins, it is vital to establish a methodological workflow to identify any microcystin in a complex sample, regardless of the availability of a reference standard. Additionally, ascribing varying levels of confidence to these identifications is critical to effectively communicate discoveries. MethodsA liquid-chromatography–high-resolution mass spectrometry method was utilized to identify microcystins present in cyanobacterial extracts from a strain of Microcystis aeruginosa and an Aphanizomenon sp. First, microcystin congeners with available standards were identified in the cyanobacterial extract. These known-unknown microcystins were considered to have the highest confidence identifications due to availability of accurate masses, retention times, and library spectra for comparison. Utilizing the spectra of these microcystins, relatively high-abundance diagnostic product-ions were identified and employed to screen the data for additional candidate microcystins. Microcystins without a standard that had an exact mass matching a microcystin published in CyanoMetDB were considered semi-known-unknown microcystins. The remaining microcystins were considered unknown-unknown microcystins. The identities of the microcystins determined herein were additionally supported by product-ion analysis, thiol reactivity, esterification reactions, neutral loss analysis, and literature contextualization. ResultsIn total, utilizing the systematic workflow presented herein, 23 microcystins were identified in the M. aeruginosa culture, including two not published previously: [d-Asp3]MC-LCit and the incompletely identified MC-L(C7H11NO3).

Synthesis of α-Carbonyl-α'-sulfenyl Sulfoxonium Ylides in Water at Room Temperature.

An efficient synthesis of α-carbonyl-α'-sulfenyl sulfoxonium ylides through a KIO3-promoted cross-dehydrogenative coupling reaction of aryl thiols and α-carbonyl sulfoxonium ylides in an aqueous medium at room temperature has been described. The α-carbonyl sulfoxonium ylides and aryl thiols adorned with various functional groups were well-tolerated and afforded moderate to high yields of α-carbonyl-α'-sulfenyl sulfoxonium ylide derivatives. Finally, by converting synthesized ylide 3a into other valuable compounds, we demonstrated the practicality of this synthetic method.

Oxidation mechanism of sulfur-containing compounds and antioxidant depletion dynamics: Insights into interactions

Sulfur-containing compounds (SCCs) are the most abundant heteroatomic organic species (HOS) in petroleum fuels. This study investigates the dynamics of SCCs in surrogate fuel systems, focusing on their oxidation stability, reactivity, and interactions. An analysis of eleven selected SCCs, spanning both reactive (thiols, sulfides, and disulfides) and nonreactive (thiophenes and benzothiazole) species, was conducted at 140 °C. The investigation delves into reaction and degradation mechanisms, exploring resulting products, binary mixtures of reactive SCCs for potential interactions, and multicomponent SCC systems to understand nuanced interaction. Findings reveal unique reaction pathways in single-component SCCs, synergetic and antagonistic effects in binary mixtures, and substantial decreases in reaction rates in all multicomponent systems. Moreover, SCCs expedited antioxidant consumption, highlighting their pivotal role in fuel stability and degradation. The study contributes valuable insights into SCC dynamics, advocating further exploration of HOS’s interactions for enhanced fuel formulation and stability testing strategies.

In Vitro Characterization of Kitasetaline Biosynthesis Reveals a Bifunctional P450 Decarboxylase and a Vinyl β-Carboline Intermediate Susceptible to Nonenzymatic Thiol Addition.

Kitasetaline is one of the very few β-carbolines isolated from bacteria. It features a unique N-acetylcysteine moiety linked to the β-carboline core through a thioether bond. While earlier in vivo experiments identified the gene cluster and reported several putative biosynthetic intermediates, how the C-S bond linkage is constructed has remained elusive. Herein, in vitro reconstitution of kitasetaline biosynthesis reveals the involvement of a Pictet-Spenglerase (KslB) and a promiscuous dehydrogenase (KslA) that generate the characteristic β-carboline ring system. In addition, the P450 enzyme KslC was found to catalyze oxidative decarboxylation of 1-(2-carboxyethyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid to yield the biosynthetic intermediate 1-vinyl-9H-pyrido[3,4-b]indole-3-carboxylic acid. KslC is also capable of catalyzing further oxidation of its product to yield an N-hydroxylated side product. Importantly, the vinyl intermediate was found to undergo nonenzymatic nucleophilic addition by N-acetyl-l-cysteine to generate the C-S bond leading directly to kitasetaline without the involvement of a mycothiolated intermediate proposed in a previous biosynthetic model. Thus, this work not only demonstrates that biosynthesis of β-carboline compounds is rich in unexpected chemistry but also adds to the growing realization that biological thiolation reactions are often nonenzymatic in nature, relying instead on enzymatic formation of reactive electrophiles.

Synthesis, Docking Study, and Antimicrobial Activity of Some New Heterocyclic Compounds Bearing Naphthalene Moiety

AbstractUpon reaction with α,β‐unsaturated nitriles (2 a–d) and/or salicylaldehyde, 3‐(naphthalen‐2‐yl)‐3‐oxopropanenitrile (1) yields pyridine derivatives 3 a–d and/or the imino chromene derivative 4. Compound 1 reacted with phenyl isothiocyanate in a basic medium to give the intermediate potassium salt 6. Upon stirring at room temperature, the intermediate 6 with some α‐halogenated compounds yielded the acyclic compounds 7, 9, 11, 13, and 15. On the other hand, refluxing intermediate 6 with the same α‐halogenated compounds in DMF in the presence of TEA afforded the corresponding thiophene derivatives 8, 10, 12, 14, and 16. The reaction of thiol 17 with hydrazine hydrate derivatives 18 a–d yielded the corresponding pyrazole derivatives 21 a–d. Compound 20 reacted with o‐phenylenediamine or o‐aminothiophenol in the presence of DMF/TEA to give the corresponding benzothiazole or benzoimidazole derivatives 27 a and 27 b, respectively. Thirty new synthesized compounds were examined for their antibacterial activity against Gram‐positive bacteria including S. aureus and B. subtilis, Gram‐negative bacteria such as P. aeruginosa and E. coli, and yeast‐like fungi like C. albicans in vitro. Compounds 15, 16, 21 d, 27 a, 27 b, exhibited the highest antibacterial activity against Gram‐positive bacteria. Molecular docking experiments were conducted to explore the binding affinities and interaction modalities of selected derivatives with the target PDB‐3cmt protein.

The perfluoroalkylthiolation reaction of thiols with perfluoroalkanesulfenic acids

The perfluoroalkylthiolation reactions of thiols with perfluoroalkanesulfenic acids were achieved without adding any additives, giving a series of unsymmetric disulfides in good to excellent yields. After simple removal of solvent from reaction system under reduced pressure, most products could be obtained with high purity. The reaction tolerates both aryl and alkyl thiols and has advantages such as easy-to-handle, additive-free and simple separation procedure.

Dual functional bio-inspired additives reconstruct metal-organic interface stability for wearable energy storage system

Aqueous zinc-ion batteries (AZIBs) hold significant promise across wearable energy storage devices due to their superior safety and cost-effectiveness. However, the instability of the electrode/electrolyte interface leads to severe hydrogen evolution reaction (HER) and dendritic growth. Herein, a bio-inspired additive, keratin (Ker), is introduced to construct a robust interfacial layer. The hydrogen bonding donors homogenize the distribution of interfacial hydrogen bond networks and increases the electron density in Zn2+-OH interaction, stabilizing bound H2O molecules and effectively inhibiting HER. Additionally, electron transfer from the highly reactive thiol (-SH) motivates the formation of Zn-S chemical linkage, enhancing interface stability. These features endow Zn||Zn symmetric cells with an impressive cycle life, reaching up to 3364 h at 1 mA cm−2/1 mAh cm−2. The Zn||α-MnO2 and Zn||VO2 full cells also exhibit a substantial capacity retention after 1000 cycles. Incorporating multi-step laser cutting technology enables the fabrication of flexible, large-area micro-batteries with prolonged cycle life. This study presents an advanced methodology for heralding a promising pathway to improve the reversibility of aqueous energy storage systems.

Efficient unsymmetric disulfide formation by molecular-scale tailoring of ortho-polyquinone-based polymer photocatalyst

Disulfide bonds, especially unsymmetric disulfide bonds, have important applications in bioactivity and drug molecules, but the synthesis of unsymmetric disulfide bonds remains challenging due to efficiency and selectivity issues. Herein, this work utilizes anthraquinone (AQ) and cyclictriphosphonononitrile through a nucleophilic substitution reaction to synthesize an organic polymer (ANTH-AMI) that incorporates an ortho-polyquinone (o-polyquinone) redox center. The anthraquinone molecule functions as a redox center, capable of accepting photoinduced electrons and subsequently transferring them to initiate an electron-coupled hydrogenation reaction (AQ to AQH). Moreover, the proximity of the o-polyquinone redox sites facilitates the catalysis of unsymmetric disulfide bond formation. Consequently, the ANTH-AMI photocatalysts demonstrate exceptional yields (up to 82 %), substrate versatility, cycling stability, and scalable preparation in promoting unsymmetric coupling reactions of thiol. This work offers a solution for designing organic polymer photocatalysts with adjacent multiple redox centers for cross-coupling reactions.

Comparing thiol and selenol reactivity towards peroxynitrite by computer simulation

Peroxynitrite is a very reactive species implicated in a variety of pathophysiological cellular processes. Particularly, peroxynitrite-mediated oxidation of cellular thiol-containing compounds such as cysteine residues is a key process which has been extensively studied. Cysteine plays roles in many redox biochemistry pathways. In contrast, selenocysteine, the 21st amino acid, is only present in 25 human proteins. Investigating the molecular basis of selenocysteine's reactivity may provide insights into its unique role in these selenocysteine-containing proteins. The two-electron oxidation of thiols or selenols by peroxynitrite is a process that is carried out by the thiolate/selenate forms and peroxynitrous acid.In this work, we shed light on the molecular basis of the differential reactivity of both species towards peroxynitrite by means of state-of-the-art computer simulations. We performed electronic structure calculations of the reaction in the methanethiolate and methaneselenolate model systems with peroxynitrous acid at different levels of theory using an implicit solvent scheme. In addition, we employed a multi-scale quantum mechanics/molecular mechanics approach for obtaining free energy profiles of these chemical reactions in aqueous solution, which enabled the comparison between the simulations and the available experimental data. Our results suggest that the larger reactivity observed in the selenocysteine case at physiological pH is mainly due to the lower pKa, which affords a larger fraction of the reactive anionic species in these conditions, and in a second place to a slightly enhanced intrinsic reactivity of the selenate form due to its larger nucleophilicity.

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.

A Combination of Cardamonin and Doxorubicin Selectively Affect Cell Viability of Melanoma Cells: An In Vitro Study.

Treatment of the most aggressive and deadliest form of skin cancer, the malignant melanoma, still has room for improvement. Its invasive nature and ability to rapidly metastasize and to develop resistance to standard treatment often result in a poor prognosis. While the highly effective standard chemotherapeutic agent doxorubicin (DOX) is widely used in a variety of cancers, systemic side effects still limit therapy. Especially, DOX-induced cardiotoxicity remains a big challenge. In contrast, the natural chalcone cardamonin (CD) has been shown to selectively kill tumor cells. Besides its anti-tumor activity, CD exhibits anti-oxidative, anti-inflammatory and anti-bacterial properties. In this study, we investigated the effect of the combinational treatment of DOX with CD on A375 melanoma cells compared to normal human dermal fibroblasts (NHDF) and rat cardiac myoblasts (H9C2 cells). DOX-induced cytotoxicity was unselective and affected all cell types, especially H9C2 cardiac myoblasts, demonstrating its cardiotoxic effect. In contrast, CD only decreased the cell viability of A375 melanoma cells, without harming normal (healthy) cells. The addition of CD selectively protected human dermal fibroblasts and rat cardiac myoblasts from DOX-induced cytotoxicity. While no apoptosis was induced by the combinational treatment in normal (healthy) cells, an apoptosis-mediated cytotoxicity was demonstrated in A375 melanoma cells. CD exhibited thiol reactivity as it was able to directly interact with N-acetylcysteine (NAC) in a cell-free assay and to induce heme oxygenase-1 (HO-1) in all cell types. And that took place in a reactive oxygen species (ROS)-independent manner. DOX decreased the mitochondrial membrane potential (Δψm) in all cell types, whereas CD selectively decreased mitochondrial respiration, affecting basal respiration, maximal respiration, spare respiratory capacity and ATP production in A375 melanoma cells, but not in healthy cardiac myoblasts. The DOX-induced cytotoxicity seen in melanoma cells was ROS-independent, whereas the cytotoxic effect of CD was associated with CD-induced ROS-formation and/or its thiol reactivity. This study highlights the beneficial properties of the addition of CD to DOX treatment, which might protect patients from DOX-induced cardiotoxicity. Future experiments with other tumor cell lines or a mouse model should substantiate this hypothesis.

On-Demand Thio-Succinimide Hydrolysis for the Assembly of Stable Protein-Protein Conjugates.

Chemical post-translational protein-protein conjugation is an important technique with growing applications in biotechnology and pharmaceutical research. Maleimides represent one of the most widely employed bioconjugation reagents. However, challenges associated with the instability of first- and second-generation maleimide technologies are yet to be fully addressed. We report the development of a novel class of maleimide reagents that can undergo on-demand ring-opening hydrolysis of the resulting thio-succinimide. This strategy enables rapid post-translational assembly of protein-protein conjugates. Thio-succinimide hydrolysis, triggered upon application of chemical, photochemical, or enzymatic stimuli, allowed homobifunctional bis-maleimide reagents to be applied in the production of stable protein-protein conjugates, with complete temporal control. Bivalent and bispecific protein-protein dimers constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce protein-protein-fluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable protein-protein conjugates.

Preferential Secretion of Oxidation-Sensitive Proteins by Unconventional Pathways: Why is This Important for Inflammation?

Significance: Fidelity of intercellular communication depends on unambiguous interactions between protein ligands and membrane receptors. Most proteins destined to the extracellular space adopt the required three-dimensional shape as they travel through the endoplasmic reticulum (ER), Golgi complex, and other organelles of the exocytic pathway. However, some proteins, many of which are involved in inflammation, avoid this classical secretory route and follow unconventional pathways to leave the cell. Recent Advances: Stringent quality control systems operate in the ER and cis-Golgi, restricting transport to native conformers, devoid of non-native disulfides and/or reactive thiols. However, some proteins released by living cells require reduced cysteines to exert their extracellular function(s). Remarkably, these proteins lack the secretory signal sequence normally required by secretory proteins for translocation into the ER lumen. Critical Issues: Why do interleukin-1β, high mobility group box 1, and other proinflammatory proteins avoid the ER-Golgi route to reach the intercellular space? These proteins require reactive cysteines for exerting their function. Therefore, eluding thiol-mediated quality control along the exocytic pathway is likely one of the main reasons why extracellular proteins that need to be reduced utilize unconventional pathways of secretion, where a quality control aimed at oxidating native cysteines is not present. Future Directions: Particularly under stress conditions, cells release redox-active enzymes and nonprotein thiol compounds that exert an extracellular control of redox-sensitive protein activity, shaping inflammatory responses. This post-secretion, redox-dependent editing of protein messages is still largely undefined. Understanding the underlying mechanistic events will hopefully provide new tools to control inflammation. Antioxid. Redox Signal. 41, 693-705.

Visible-Light-Mediated gem-Difunctionalization of Diazo Compounds with Vinyl Sulfoxonium Ylides and Thiols

AbstractA visible-light-induced multicomponent reaction of vinyl sulfoxonium ylide, thiol, and diazo ester to generate tertiary sulfide is described. The present diastereoselective gem-difunctionalization of the diazo ester can be achieved under mild conditions, as it does not require any additives, catalysts, or transition metals and is tolerant of air and moisture. Due to more nucleophilicity, vinyl sulfoxonium ylide undergoes S–H insertion with thiols to generate an allyl sulfide intermediate. Simultaneously, diazo ester undergoes photolysis to generate a carbene intermediate. Subsequently, the coupling of carbene and allyl sulfide intermediates generates sulfonium ylide, which undergoes Doyle–Kirmse rearrangement to generate tertiary sulfide scaffolds.

Hemoglobin’s β-subunit is primed to synergize oxygen delivery with nitric oxide-mediated increased blood flow

Hemoglobin (Hb) undergoes a well-documented R[Formula: see text]T quaternary transition from a high [Formula: see text] affinity R state to a low [Formula: see text] affinity T state to optimize [Formula: see text] delivery to tissues. Also, red blood cells (RBCs) release a nitrovasodilator that increases blood flow to boost [Formula: see text] delivery. Hb’s R[Formula: see text]T transition coordinates its [Formula: see text] desaturation and RBC nitrovasodilator release by much-debated mechanisms. Here we investigate the allosterically controlled [Formula: see text]-nitrosation of Hb at its conserved βCys93 (i.e., SNO-Hb formation) for nitrovasodilator release. First, we examined NO[Formula: see text]/deoxyHb (HbFeII) incubations following aeration to mimic RBC NO production by the nitrite reductase activity of HbFeII and trapping of the nascent NO by βCys93 to give SNO-Hb on HbFeII conversion to oxyHb (HbFeO2). We confirmed SNO-Hb formation in the incubations with yields modulated by RBC antioxidant enzymes and [Formula: see text] but not CO. Since FeO2 hemes scavenge free NO, we hypothesized NO channeling within Hb and found by molecular dynamics simulations that most unligated NO molecules placed in the β-distal heme pocket (βDP) rapidly diffuse into a wide β-tunnel connecting the βDP to Hb’s central cavity and βCys93. Contraction of the central cavity brings NO closer to βCys93 in R-state plus βPhe71 and βTyr145 adopt conformations favorable to thiol access and SNO-Hb formation. In T-state, the SNO group is surface-exposed and destabilized to extrude NO. Thus, its structure, dynamics and conserved reactive thiol (βCys93) suggest that the β-subunit evolved to synergize [Formula: see text] and nitrovasoactivity delivery to tissues as a function of Hb [Formula: see text] saturation.