S-nitrosothiols as nitrite alternatives: Effects on residual nitrite, lipid oxidation, volatile profile, and cured color of restructured cooked ham.

This study evaluated the use of the S-nitrosothiols, S-nitroso-N-acetylcysteine (NAC-SNO) and S-nitroso-N-acetylcysteine ethyl ester (NACET-SNO), at different concentrations (25-300 mg nitrite equivalent - NEq/kg) as sodium nitrite substitutes in restructured cooked hams. The pH value and instrumental cured color were not affected by the type or amount of curing agent used. Products with 25 and 50 mg/kg ingoing nitrite had lower thiobarbituric acid-reactive substance values than those with equimolar amounts of S-nitrosothiols. Products with >150 mg NEq/kg of S-nitrosothiols had residual nitrite similar to 50 mg/kg nitrite, and this resulted in the same volatile compound profile as nitrite added in equimolar amounts. A 300 mg NEq/kg of S-nitrosothiols was required to obtain a similar and minimally stable cured pink color perception as sliced samples with 50-150 mg/kg added nitrite. The results obtained reinforce the great potential of both alternative curing agents in the complete replacement of nitrite by equimolar amounts in restructured cooked products; however, differences in cured color stability should be considered.

Identification of Protein Targets of S-Nitroso-Coenzyme A-Mediated S-Nitrosation Using Chemoproteomics.

S-Nitrosation is a cysteine post-translational modification fundamental to cellular signaling. This modification regulates protein function in numerous biological processes in the nervous, cardiovascular, and immune systems. Small molecule or protein nitrosothiols act as mediators of NO signaling by transferring the NO group (formally NO+) to a free thiol on a target protein through a transnitrosation reaction. The protein targets of specific transnitrosating agents and the extent and functional effects of S-nitrosation on these target proteins have been poorly characterized. S-nitroso-coenzyme A (CoA-SNO) was recently identified as a mediator of endogenous S-nitrosation. Here, we identified direct protein targets of CoA-SNO-mediated transnitrosation using a competitive chemical-proteomic approach that quantified the extent of modification on 789 cysteine residues in response to CoA-SNO. A subset of cysteines displayed high susceptibility to modification by CoA-SNO, including previously uncharacterized sites of S-nitrosation. We further validated and functionally characterized the functional effects of S-nitrosation on the protein targets phosphofructokinase (platelet type), ATP citrate synthase, and ornithine aminotransferase.

Promotion of S-nitrosation of cysteine by a {Co(NO)2}10 complex.

S-Nitrosothiols (SNOs) serve as endogenous carriers and donors of NO within living cells, releasing nitrosonium ions (NO+), NO, or other nitroso derivatives. In this study, we present a bioinspired {Co(NO)2}10 complex 1 that achieved S-nitrosation towards Cys residues. The incorporation of a ferrocenyl group in 1 allowed for fine-tuning of the nitrosation reaction, taking advantage of the redox ability of Cys residues. Complex 1 was synthesized and characterized, demonstrating its NO translation reactivity. Furthermore, complex 1 successfully converted Cys into S-nitrosocysteine (Cys-SNO), as confirmed by UV-Vis, IR, and XAS spectroscopy. This study presents a promising approach for S-nitrosation of Cys residues for further exploration in the modification of Cys-containing peptides.

GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using 15N and 18O Isotopes.

Nitrite (O=N-O-, NO2-) and nitrate (O=N(O)-O-, NO3-) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (●NO). ●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ●NO synthases. It is considered that the autoxidation of ●NO in aqueous solutions and in O2-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O- + H+; pKaHONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (14NO2-) and labeled nitrite (15NO2-) and RSNO (RS15NO, RS15N18O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H216O) or labeled H2O (H218O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography-mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl2 accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating 18O from H218O into the SNO group. In aqueous buffers prepared in H218O, synthetic peroxynitrite (ONOO-) decomposes to nitrite without 18O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS15NO and H218O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ●NO and hydrolysis of RSNO.

Unraveling the Molecular Mechanism of S-Nitrosation Mediated by N-Acetylmicroperoxidase-11.

Conversion of NO to stable S-nitrosothiols is perceived as a biologically important strategy of NO storage and a signal transduction mechanism. Transition-metal ions and metalloproteins are competent electron acceptors that may promote the formation of S-nitrosothiols from NO. We selected N-acetylmicroperoxidase (AcMP-11), a model of protein heme centers, to study NO incorporation to three biologically relevant thiols (glutathione, cysteine, and N-acetylcysteine). The efficient formation of S-nitrosothiols under anaerobic conditions was confirmed with spectrofluorimetric and electrochemical assays. AcMP-11-assisted incorporation of NO to thiols occurs via an intermediate characterized as an N-coordinated S-nitrosothiol, (AcMP-11)Fe2+(N(O)SR), which is efficiently converted to (AcMP-11)Fe2+(NO) in the presence of NO excess. Two possible mechanisms of S-nitrosothiol formation at the heme-iron were considered: a nucleophilic attack on (AcMP-11)Fe2+(NO+) by a thiolate and a reaction of (AcMP-11)Fe3+(RS) with NO. Kinetic studies, performed under anaerobic conditions, revealed that the reversible formation of (AcMP-11)Fe2+(N(O)SR) occurs in a reaction of RS- with (AcMP-11)Fe2+(NO+) and excluded the second mechanism, indicating that the formation of (AcMP-11)Fe3+(RS) is a dead-end equilibrium. Theoretical calculations revealed that N-coordination of RSNO to iron, forming (AcMP-11)Fe2+(N(O)SR), shortens the S-N bond and increases the complex stability compared to S-coordination. Our work unravels the molecular mechanism of heme-iron-assisted interconversion of NO and low-molecular-weight thiols to S-nitrosothiols and recognizes the reversible NO binding in the form of a heme-Fe2+(N(O)SR) motif as an important biological strategy of NO storage.

Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo.

S-nitrosothiols are labile thiol-NO adducts formed in vivo primarily by metalloproteins such as NO synthase, ceruloplasmin, and hemoglobin. Abnormal S-nitrosothiol synthesis and catabolism contribute to many diseases, ranging from asthma to septic shock. Current methods for quantifying S-nitrosothiols in vivo are suboptimal. Samples need to be removed from the body for analysis, and the S-nitrosothiols can be broken down during ex vivo processing. Here, we have developed a noninvasive device to measure mammalian tissue S-nitrosothiols in situ non-invasively using ultraviolet (UV) light, which causes NO release in proportion to the S-nitrosothiol concentration. We validated the assay in vitro; then, we applied it to measure S-nitrosothiols in vivo in rats and in humans. The method was sensitive to 0.5 µM, specific (did not detect other nitrogen oxides), and was reproducible in rats and in humans. This noninvasive approach to S-nitrosothiol measurements may be applicable for use in human diseases.

Mechanisms of nitric oxide generation in living systems.

In modern chemical and biochemical studies, special attention is paid to molecular systems capable of generating nitric oxide (NO), which is one of the most important signalling molecules in the body and can trigger a whole cascade of reactions. Despite the importance of this molecule, the mechanisms of its formation in living organisms remain a subject of debate. This review combines the most important methods of releasing NO from endogenous and exogenous sources. The history of endogenous NO donors dates back more than 150 years, since the synthesis of nitroglycerin, which remains the standard vasodilator today, even though it is known that it and many other similar compounds lead to the development of a nitrate tolerance. Particular awareness is devoted to the mechanisms of NO formation without the participation of enzymes, since these methods are most important for creating exogenous sources of NO as drugs. The study of NO formation methods is centred on both the creation of new NO donors and understanding the mechanisms of tolerance to them.

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates.

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1-10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Buffer concentration dramatically affects the stability of S-nitrosothiols in aqueous solutions.

S-nitrosothiols (RSNOs) are an important group of nitric oxide (NO)-donating compounds with low toxicity and wide biomedical applications. In this paper, we, for the first time, demonstrate that the concentration of buffer remarkably affects the stability of RSNOs including naturally occurring S-nitrosoglutathione (GSNO) and synthetic S-nitroso-N-acetylpenicillamine (SNAP). For a solution with a high concentration of GSNO (e.g., 50 mM) and an initial near-neutral pH, the optimal buffer concentration is close to the GSNO concentration under our experimental conditions. A lower buffer concentration does not have adequate buffer capacity to resist the pH drop caused by GSNO decomposition. The decreased solution pH further accelerates GSNO decomposition because GSNO is most stable at near-neutral pH according to our density functional theory (DFT) calculations. A higher-than-optimal buffer concentration also reduces the GSNO stability because buffer ingredients including phosphate, Tris base, and HEPES consume NO/N2O3. In contrast to GSNO, the highest SNAP stability is obtained when the starting solution at a neutral pH does not contain buffer species, and the stability decreases as the buffer concentration increases. This is because SNAP is more stable at mildly acidic pH and the SNAP decomposition-induced pH drop stabilizes the donor. When the RSNO concentration is low (e.g., 1 mM), the buffer concentration also matters because any excess buffer accelerates the donor decomposition. Since the effect of buffer concentration was previously overlooked and suboptimal buffer concentrations were often used, this paper will aid in the formulation of RSNO solutions to obtain the maximum stability for prolonged storage and sustained NO release.

Stability of S-nitrosothiols and S-nitrosylated proteins: A struggle for cellular existence!

Nitric oxide is a well-known gasotransmitter molecule that covalently docks to sulfhydryl groups of proteins resulting in S-nitrosylation of proteins and nonprotein thiols that serve a variety of cellular processes including cGMP signaling, vasodilatation, neurotransmission, ion-channel modulation, and cardiac signaling. S-nitrosylation is an indispensable modification like phosphorylation that directly regulates the functionality of numerous proteins. However, recently there has been a controversy over the stability of S-nitrosylated proteins (PSNOs) within the cell. It has been argued that PSNOs formed within the cell is a transient intermediate step to more stable disulfide formation and disulfides are the predominant end effector modifications in NO-mediated signaling. The present article accumulates state-of-the-art evidence from numerous research that strongly supports the very existence of PSNOs within the cell and attempts to put an end to the controversy. This review illustrates critical points including comparative bond dissociation energies of S-NO bond, the half-life of S-nitrosothiols and PSNOs, cellular concentrations of PSNOs, X ray crystallographic studies on PSNOs, and stability of PSNOs at physiological concentration of antioxidants. These logical evidence cumulatively support the endogenous stability and inevitable existence of PSNOs/RSNOs within the cell that directly regulate the functionality of proteins and provide valuable insight into understanding stable S-nitrosylation mediated cell signaling.

Iron(II/III) Halide Complexes Promote the Interconversion of Nitric Oxide and S-Nitrosothiols through Reversible Fe-S Interaction.

Heme and non-heme iron in biology mediate the storage/release of NO• from S-nitrosothiols as a means to control the biological concentration of NO•. Despite their importance in many physiological processes, the mechanisms of N-S bond formation/cleavage at Fe centers have been controversial. Herein, we report the interconversion of NO• and S-nitrosothiols mediated by FeII/FeIII chloride complexes. The reaction of 2 equiv of S-nitrosothiol (Ph3CSNO) with [Cl6FeII2]2- results in facile release of NO• and formation of iron(III) halothiolate. Detailed spectroscopic studies, including in situ UV-vis, IR, and Mössbauer spectroscopy, support the interaction of the S atom with the FeII center. This is in contrast to the proposed mechanism of NO• release from the well-studied "red product" κ1-N bound S-nitrosothiol FeII complex, [(CN)5Fe(κ1-N-RSNO)]3-. Additionally, FeIII chloride can mediate NO• storage through the formation of S-nitrosothiols. Treatment of iron(III) halothiolate with 2 equiv of NO• regenerates Ph3CSNO with the FeII source trapped as the S = 3/2 {FeNO}7 species [Cl3FeNO]-, which is inert toward further coordination and activation of S-nitrosothiols. Our work demonstrates how labile iron can mediate the interconversion of NO•/thiolate and S-nitrosothiol, which has important implications toward how Nature manages the biological concentration of free NO•.

S-nitrosylated TDP-43 triggers aggregation, cell-to-cell spread, and neurotoxicity in hiPSCs and in vivo models of ALS/FTD.

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of "sporadic" cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface.

Nitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysß93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including L-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0-10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH ↔ ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.

Copper-doped metal-organic frameworks for the controlled generation of nitric oxide from endogenous S-nitrosothiols.

Nitric oxide (NO) is an essential signaling molecule with a number of biological functions and holds great promise in biomedical applications. However, NO delivery technologies have been complicated due to the inherent properties of NO which include short half-life and limited transport distance in human tissues. In addition, the biofunctionality of NO is strongly dependent on its concentrations and locations where it is delivered. To achieve controlled NO delivery, many studies have focused on encapsulating NO donors into macromolecular scaffolds or using catalysts to realize in situ NO generation from NO prodrugs. Successful applications have been shown, however NO donor-loaded platforms experience the limitation of finite NO storage capacity. The present study reports the synthesis of a catalyst, copper-doped zeolitic imidazolate framework ZIF-8 (Cu2+/ZIF-8), that is designed to generate NO from naturally occurring endogenous NO donors. By tuning the copper doping percentages, we achieved controlled NO generation from S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO). Cu2+/ZIF-8 particles retained their catalytic potency after 5 NO generation cycles and we showed that our copper-doped ZIF-8 catalyst produced a 10-fold increased amount of NO compared with previous reports. As a proof-of-concept study, we demonstrated the ability of copper-doped ZIF-8 to disperse bacterial biofilms in the presence of GSNO.

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.

Ag@S-nitrosothiol core-shell nanoparticles for chemo and photothermal synergistic tumor targeted therapy.

Along with the development of controlled delivery systems for targeted therapy, 'single-strategy' therapy often fails to achieve the desired performance in real body internal environments. In such a case, it is necessary to develop synergistic therapy strategies. Herein, for the first time, we designed and synthesized hyaluronic acid (HA) modified Ag@S-nitrosothiol core-shell nanoparticles for synergistic tumor cell targeted therapy based on photothermal therapy (PTT) and nitric oxide (NO) based chemotherapy. Triggered by near-infrared irradiation (NIR), the Ag core nanoparticle would convert the light to cytotoxic heat via a surface plasmon resonance mechanism for cancer cell apoptosis. Meanwhile, responding to NIR as well as the generated heat, the S-nitrosothiol polymeric shells would give off free NO at high concentration, inducing NO based chemotherapy. Tumor cell selective cytotoxicity assay in vitro as well as tumor bearing mouse experiments in vivo demonstrated the effective photothermal and NO based chemical synergistic tumor targeted therapy. This spatiotemporally controllable system could provide a new option and era for tumor targeted therapy in the future.

S-nitrosothiols loaded mini-sized Au@silica nanorod elicits collagen depletion and mitochondrial damage in solid tumor treatment.

To a large extent, the dense extracellular matrix (ECM), which tightly connects tumor cells to arm the tumor into an intractable fortress, significantly decreases the nanoparticles delivery efficacy and overall performance in cancer treatments. Therefore, it is necessary to transform the dense stroma of solid tumors to loose state, which could realize deep penetration of nanomedicine and enhance cancer treatment effects. Here, we fabricated a protein-free collagen nanosweeper, triphenylphosphonium bromide (TPP) coated and S-nitrosothiols loaded mini-sized Au@silica nanorod (Au@SiO2-SNO/PEG/TPP, GSNP-TPP), to clear the transport barriers of nanoparticles as well as elevate enhanced permeability and retention (EPR) effect, thus alleviating the diffusion resistance and realizing further penetration of nanoparticles. Methods: By modifying the Au@silica with thermo-sensitive S-nitrosothiols, the carrier could release the nitric oxide (NO) due to the surface overheat as well as perform photothermal therapy (PTT) under near-infrared (NIR) laser irradiation. The level of collagen depletion was observed via western blotting and immunofluorescent staining. In addition, the dual-imaging and antitumor efficiency of GSNP-TPPs were evaluated with the HeLa tumor-bearing mouse model. Results: On one hand, the released NO could deplete collagen by activating matrix metalloproteinases (MMPs) to break collagen fibers, thus loosening the dense ECM to enhance the cellular internalization. On the other hand, with the mitochondrial-targeted effect of TPP, the diffusible NO in tumor might rapidly interact with superoxide anion (O2Y-) to produce highly toxic and powerful reactive nitrogen species (RNS) -- peroxynitrite (ONOO-), which resulted in mitochondrial damage to induce cell apoptosis. With the unique properties of mini-sized gold nanorods, the formulated nanoparticles exhibited good computed tomography (CT) and multi-spectral optoacoustic tomography (MSOT) imaging effects in precisely locating and monitoring tumor. Moreover, the antitumor efficacy of GSNP-TPPs + laser group was further confirmed by ex-vivo histological analysis of tumor tissue. Conclusion: This work points out a strategy to overcome the obstacle standing in nanoparticles penetration, and opens the door of further exploitation of NO-related theranostic systems.

Nanosized copper(ii) oxide/silica for catalytic generation of nitric oxide from S-nitrosothiols.

Nitric oxide NO, mediates inflammatory and thrombotic processes and designing biomaterials capable of releasing NO in contact with biological tissues is considered to be a major factor aimed at improving their bio- and haemocompatibility and antibacterial properties. Their NO-releasing capacity however is limited by the amount of the NO-containing substance incorporated in the bulk or immobilised on the surface of a biomaterial. An alternative approach is based on the design of a material generating nitric oxide from endogenous NO bearing metabolites by their catalytic decomposition. It offers, at least in theory, an unlimited source of NO for as long as the material remains in contact with blood and the catalyst maintains its activity. In this paper we studied the catalytic properties of novel nanostructured CuO/SiO2 catalysts in generating NO by decomposition of S-nitrosoglutathione (GSNO) in vitro. CuO/SiO2 catalysts with different CuO loadings were synthesized by chemisorption of copper(ii) acetylacetonate on fumed nanosilica followed by calcination. CuO content was controlled by a number of chemisorption-calcination cycles. Fourier-transform infrared spectroscopy and thermogravimetric analysis confirmed the formation of CuO/SiO2 nanoparticles (NPs) with particle size of CuO phase in the range from 71 to 88 nm. Scanning electron microscopy images revealed a uniform distribution of NPs without their sintering or agglomeration. All the materials of the CuO/SiO2 NP series exhibited NO-generating activity from GSNO confirmed by the Griess assay and by measuring the concentration of nitrite and nitrate anions in model solutions such as phosphate buffered saline and bovine serum. This activity is dependent on the material specific surface area and CuO exposure on the surface rather than CuO bulk content. The rate of NO production increased at higher initial concentration of the NO-bearing substrate studied in the range between 0.01 mM and 1.0 mM RSNO, which covers its physiological level. CuO/SiO2 NPs can be used to design polymers with NO generating properties at blood-biomaterial interface which are expected to have improved biocompatibility thus enhancing their potential for medical applications such as surgical tubing, peripheral venous catheters, auxiliary blood circulation devices and drug-eluting balloons.

Biological control of S-nitrosothiol reactivity: potential role of sigma-hole interactions.

S-Nitrosothiols (RSNOs) are ubiquitous biomolecules whose chemistry is tightly controlled in vivo, although the specific molecular mechanisms behind this biological control remain unknown. In this work, we demonstrate, using high-level ab initio and DFT calculations, the ability of RSNOs to participate in intermolecular interactions with electron pair donors/Lewis bases (LBs) via a σ-hole, a region of positive electrostatic potential on the molecular surface at the extension of the N-S bond. Importantly, σ-hole binding is able to modulate the properties of RSNOs by changing the balance between two chemically opposite (antagonistic) resonance components, R-S+[double bond, length as m-dash]N-O- (D) and R-S-/NO+ (I), which are, in addition to the main resonance structure R-S-N[double bond, length as m-dash]O, necessary to describe the unusual electronic structure of RSNOs. σ-Hole binding at the sulfur atom of RSNO promotes the resonance structure D and reduces the resonance structure I, thereby stabilizing the weak N-S bond and making the sulfur atom more electrophilic. On the other hand, increasing the D-character of RSNO by other means (e.g. via N- or O-coordination of a Lewis acid) in turn enhances the σ-hole bonding. Our calculations suggest that in the protein environment a combination of σ-hole bonding of a negatively charged amino acid sidechain at the sulfur atom and N- or O-coordination of a positively charged amino acid sidechain is expected to have a profound effect on the RSNO electronic structure and reactivity.

Lewis Acid Coordination Redirects S-Nitrosothiol Signaling Output.

S-Nitrosothiols (RSNOs) serve as air-stable reservoirs for nitric oxide in biology. While copper enzymes promote NO release from RSNOs by serving as Lewis acids for intramolecular electron-transfer, redox-innocent Lewis acids separate these two functions to reveal the effect of coordination on structure and reactivity. The synthetic Lewis acid B(C6 F5 )3 coordinates to the RSNO oxygen atom, leading to profound changes in the RSNO electronic structure and reactivity. Although RSNOs possess relatively negative reduction potentials, B(C6 F5 )3 coordination increases their reduction potential by over 1 V into the physiologically accessible +0.1 V vs. NHE. Outer-sphere chemical reduction gives the Lewis acid stabilized hyponitrite dianion trans-[LA-O-N=N-O-LA]2- [LA=B(C6 F5 )3 ], which releases N2 O upon acidification. Mechanistic and computational studies support initial reduction to the [RSNO-B(C6 F5 )3 ] radical anion, which is susceptible to N-N coupling prior to loss of RSSR.