Bioinspired superhydrophobic surfaces with silver and nitric oxide-releasing capabilities to prevent device-associated infections and thrombosis.

Bacteria-associated infections and thrombus formation are the two major complications plaguing the application of blood-contacting medical devices. Therefore, functionalized surfaces and drug delivery for passive and active antifouling strategies have been employed. Herein, we report the novel integration of bio-inspired superhydrophobicity with nitric oxide release to obtain a functional polymeric material with anti-thrombogenic and antimicrobial characteristics. The nitric oxide release acts as an antimicrobial agent and platelet inhibitor, while the superhydrophobic components prevent non-specific biofouling. Widely used medical-grade silicone rubber (SR) substrates that are known to be susceptible to biofilm and thrombus formation were dip-coated with fluorinated silicon dioxide (SiO2) and silver (Ag) nanoparticles (NPs) using an adhesive polymer as a binder. Thereafter, the resulting superhydrophobic (SH) SR substrates were impregnated with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) to obtain a superhydrophobic, Ag-bound, NO-releasing (SH-SiAgNO) surface. The SH-SiAgNO surfaces had the lowest amount of viable adhered E. coli (> 99.9 % reduction), S. aureus (> 99.8 % reduction), and platelets (> 96.1 % reduction) as compared to controls while demonstrating no cytotoxic effects on fibroblast cells. Thus, this innovative approach is the first to combine SNAP with an antifouling SH polymer surface that possesses the immense potential to minimize medical device-associated complications without using conventional systemic anticoagulation and antibiotic treatments.

Recent advances on the development of NO-releasing molecules (NORMs) for biomedical applications.

Nitric oxide (NO) is an important biological messenger as well as a signaling molecule that participates in a broad range of physiological events and therapeutic applications in biological systems. However, due to its very short half-life in physiological conditions, its therapeutic applications are restricted. Efforts have been made to develop an enormous number of NO-releasing molecules (NORMs) and motifs for NO delivery to the target tissues. These NORMs involve organic nitrate, nitrite, nitro compounds, transition metal nitrosyls, and several nanomaterials. The controlled release of NO from these NORMs to the specific site requires several external stimuli like light, sound, pH, heat, enzyme, etc. Herein, we have provided a comprehensive review of the biochemistry of nitric oxide, recent advancements in NO-releasing materials with the appropriate stimuli of NO release, and their biomedical applications in cancer and other disease control.

Development of an innovative turn-on fluorescent probe for targeted in-vivo detection of nitric oxide in rat brain extracts as a biomarker for migraine disease.

Nitric oxide (NO) is one of the reactive nitrogen species (RNS) that has been proposed to be a key signaling molecule in migraine. Migraine is a neurological disorder that is linked to irregular NO levels, which necessitates precise NO quantification for effective diagnosis and treatment. This work introduces a novel fluorescent probe, 2,3-diaminonaphthelene-1,4-dione (DAND), which was designed and synthesized to selectively detect NO in-vitro and in-vivo as a migraine biomarker. DAND boasts high aqueous solubility, biocompatibility, and facile synthesis, which enable highly selective and sensitive detection of NO under physiological conditions. NO reacts with diamine moieties (recognition sites) of DAND, results in the formation of a highly fluorescent product (DAND-NO) known as 1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione at λem 450 nm. The fluorescence turn-on sensing mechanism operates through an intramolecular charge transfer (ICT) mechanism. To maximize fluorescence signal intensity, parameters including DAND concentration, reaction temperature, reaction time and pH were systematically optimized for sensitive and precise NO determination. The enhanced detection capability (LOD = 0.08 µmol L-1) and high selectivity of the probe make it a promising tool for NO detection in brain tissue homogenates. This demonstrates the potential diagnostic value of the probe for individuals suffering from migraine. Furthermore, this study sheds light on the potential role of zolmitriptan (ZOLM), an antimigraine medication, in modulating NO levels in the brain of rats with nitroglycerin-induced migraine, emphasizing its significant impact on reducing NO levels. The obtained results could have significant implications for understanding how ZOLM affects NO levels and may aid in the development of more targeted and effective migraine treatment strategies.

Photostimulated Extended Nitric Oxide (NO) Release from Water-Soluble Block Copolymer to Enhance Antibacterial Activity.

N-Nitrosamines are well established motifs to release nitric oxide (NO) under photoirradiation. Herein, a series of amphiphilic N-nitrosamine-based block copolymers (BCPx-NO) are developed to attain controlled NO release under photoirradiation (365 nm, 3.71 mW/cm2). The water-soluble BCPx-NO forms micellar architecture in aqueous medium and exhibits a sustained NO release of 92-160 µM within 11.5 h, which is 36.8-64.0% of the calculated value. To understand the NO release mechanism, a small molecular NO donor (NOD) resembling the NO releasing functional motif of BCPx-NO is synthesized, which displays a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar BCPx-NO enhances antibacterial activity over NOD and exhibits a superior bactericidal effect on Gram-positive Staphylococcus aureus. In relation to biomedical applications, this work offers a comprehensive insight into tuning light-triggered NO release to improve antibacterial activity.

A Trojan horse approach for enhancing the cellular uptake of a ruthenium nitrosyl complex.

Ruthenium nitrosyl (RuNO) complexes continue to attract significant research interest due to several appealing features that make these photoactivatable nitric oxide (NO˙) donors attractive for applications in photoactivated chemotherapy. Interesting examples of molecular candidates capable of delivering cytotoxic concentrations of NO˙ in aqueous media have been discussed. Nevertheless, the question of whether most of these highly polar and relatively large molecules are efficiently incorporated by cells remains largely unanswered. In this paper, we present the synthesis and the chemical, photophysical and photochemical characterization of RuNO complexes functionalized with 17α-ethinylestradiol (EE), a semisynthetic steroidal hormone intended to act as a molecular Trojan horse for the targeted delivery of RuNO complexes. The discussion is centered around two main molecular targets, one containing EE (EE-Phtpy-RuNO) and a reference compound lacking this biological recognition fragment (Phtpy-RuNO). While both complexes displayed similar optical absorption profiles and NO˙ release efficiencies in aqueous media, important differences were found regarding their cellular uptake towards dermal fibroblasts, with EE-Phtpy-RuNO gratifyingly displaying a remarkable 10-fold increase in cellular uptake when compared to Phtpy-RuNO, thus demonstrating the potential drug-targeting capabilities of this biomimetic steroidal conjugate.

Exploring second coordination sphere effects in flavodiiron nitric oxide reductase model complexes.

Flavodiiron nitric oxide reductases (FNORs) equip pathogens with resistance to nitric oxide (NO), an important immune defense agent in mammals, allowing these pathogens to proliferate in the human body, potentially causing chronic infections. Understanding the mechanism of how FNORs mediate the reduction of NO contributes to the greater goal of developing new therapeutic approaches against drug-resistant strains. Recent density functional theory calculations suggest that a second coordination sphere (SCS) tyrosine residue provides a hydrogen bond that is critical for the reduction of NO to N2O at the active site of FNORs [J. Lu, B. Bi, W. Lai and H. Chen, Origin of Nitric Oxide Reduction Activity in Flavo-Diiron NO Reductase: Key Roles of the Second Coordination Sphere, Angew. Chem., Int. Ed., 2019, 58, 3795-3799]. Specifically, this H-bond stabilizes the hyponitrite intermediate and reduces the energetic barrier for the N-N coupling step. At the same time, the role of the Fe⋯Fe distance and its effect on the N-N coupling step has not been fully investigated. In this study, we equipped the H[BPMP] (= 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol) ligand with SCS amide groups and investigated the corresponding diiron complexes with 0-2 bridging acetate ligands. These amide groups can form hydrogen bonds with the bridging acetate ligand(s) and potentially the coordinated NO groups in these model complexes. At the same time, by changing the number of bridging acetate ligands, we can systematically vary the Fe⋯Fe distance. The reactivity of these complexes with NO was then investigated, and the formation of stable iron(II)-NO complexes was observed. Upon one-electron reduction, these NO complexes form Dinitrosyl Iron Complexes (DNICs), which were further characterized using IR and EPR spectroscopy.

Nitrite Formation at a Diiron Dinitrosyl Complex.

Pathogenic bacteria employ iron-containing enzymes to detoxify nitric oxide (NO•) produced by mammals as part of their immune response. Two classes of diiron proteins, flavodiiron nitric oxide reductases (FNORs) and the hemerythrin-like proteins from mycobacteria (HLPs), are upregulated in bacteria in response to an increased local NO• concentration. While FNORs reduce NO• to nitrous oxide (N2O), the HLPs have been found to either reduce nitrite to NO• (YtfE), or oxidize NO• to nitrite (Mka-HLP). Various structural and functional models of the diiron site in FNORs have been developed over the years. However, the NO• oxidation reactivity of Mka-HLP has yet to be replicated with a synthetic complex. Compared to the FNORs, the coordination environment of the diiron site in Mka-HLP contains one less carboxylate ligand and, therefore, is expected to be more electron-poor. Herein, we synthesized a new diiron complex that models the electron-poor coordination environment of the Mka-HLP diiron site. The diferrous precursor FeIIFeII reacts with NO• to form a diiron dinitrosyl species ({FeNO}72), which is in equilibrium with a mononitrosyl diiron species (FeII{FeNO}7) in solution. Both complexes can be isolated and fully characterized. However, only oxidation of {FeNO}72 produced nitrite in high yield (71%). Our study provides the first model that reproduces the NO• oxidase reactivity of Mka-HLP and suggests intermediacy of an {FeNO}6/{FeNO}7 species.

Ultraviolet Light Responsive N-Nitroso Polymers for Antibacterial Nitric Oxide Delivery.

This study investigates the incorporation of active secondary amine moieties into the polymer backbone by co-polymerizing 2,4,6-tris(chloromethyl)-mesitylene with three diamines, namely 1,4-diaminobutane, m-phenylenediamine, and p-phenylenediamine. This process results in the stabilization of the amine moieties and the subsequently introduced nitroso groups. Charging bioactive nitric oxide (NO) into the polymers is accomplished by converting the amine moieties into N-nitroso groups. The ability of the polymers to store and release NO depends on their structures, particularly the amount of incorporated active secondary amines. With grafting photosensitive N-nitroso groups into the polymers, the derived NO@polymers exhibit photoresponsivity. NO release is completely regulated by adjusting UV light irradiation. These resulting polymeric NO donors demonstrate remarkable bactericidal and bacteriostatic activity, effectively eradicating E. coli bacteria and inhibiting their growth. The findings from this study hold promising implications for combining NO delivery with phototherapy in various medical applications.

Supramolecular Assemblies of Fluorescent Nitric Oxide Photoreleasers with Ultrasmall Cyclodextrin Nanogels.

Developing biocompatible nitric oxide (NO) photoreleasing nanoconstucts is of great interest in view of the large variety of biological roles that NO plays and the unique advantage light offers in controlling NO release in space and time. In this contribution, we report the supramolecular assemblies of two NO photodonors (NOPDs), NBF-NO and RHD-NO, as water-dispersible nanogels, ca. 10 nm in diameter, based on γ-cyclodextrins (γ-CDng). These NOPDs, containing amino-nitro-benzofurazan and rhodamine chromophores as light harvesting antennae, can be activated by visible light, are highly hydrophobic and can be effectively entrapped within the γ-CDng. Despite being confined in a very restricted environment, neither NOPD suffer self-aggregation and preserve their photochemical and photophysical properties well. The blue light excitation of the weakly fluorescent γ-CDng/NBF-NO complex results in effective NO release and the concomitant generation of the highly green, fluorescent co-product, which acts as an optical NO reporter. Moreover, the green light excitation of the persistent red fluorescent γ-CDng/RHD-NO triggers NO photorelease without significantly modifying the emission properties. The activatable and persistent fluorescence emissions of the NOPDs are useful for monitoring their interactions with the Gram-positive methicillin-resistant Staphylococcus aureus, whose growth is significantly inhibited by γ-CDng/RHD-NO upon green light irradiation.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation.

Nitrobindins (Nbs) represent an evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum-classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

Commercial SCR catalyst modified with Cu metal to simultaneously efficiently remove NO and toluene in the fuel gas.

Developing an environmentally friendly selective catalytic reduction (SCR) catalyst to effectively eliminate both nitric oxides (NO) and toluene has garnered significant attention for regulating emissions from automobiles and the combustion of fossil fuels. This study synthesized a series of novel commercial V2O5-WO3/TiO2 catalysts modified with Cu through the wet impregnation method, which was employed to simultaneously remove NO and toluene from the fuel gas. The assessment of catalyst removal performance was conducted at a selective catalytic reduction system, and the experimental results showed a significant increase in the catalytic activity due to the modification of the copper metal. The 10% Cu/SCR catalyst showed a superior activity that the NO and toluene conversion reached 100% and 95.56% at 300 °C, respectively. Subsequently, various characterization techniques were employed to investigate the crystal phase, morphology, physical features, chemical states, and surface acidity properties of the synthesis catalysts. According to the characterization results, the presence of Cu metal did not have a noticeable impact on the physical property. However, the redox performance was enhanced, and the number of surface acidic sites was also increased after adding Cu to the SCR catalyst. Furthermore, the redox cycle of Cu metal and V species was facilitated to produce more active oxygen which helped to improve the NO and toluene conversion. This work offered a novel perspective into the synergistic oxidation of both NO and toluene, which was potentially relevant for improving the selective catalytic reduction process in coal-fired power plants.

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.

Computational Insights into the Mechanism of Nitric Oxide Generation from S-Nitrosoglutathione Catalyzed by a Copper Metal-Organic Framework.

The controlled generation of nitric oxide (NO) from endogenous sources, such as S-nitrosoglutathione (GSNO), has significant implications for biomedical implants due to the vasodilatory and other beneficial properties of NO. The water-stable metal-organic framework (MOF) Cu-1,3,5-tris[1H-1,2,3-triazol-5-yl]benzene has been shown to catalyze the production of NO and glutathione disulfide (GSSG) from GSNO in aqueous solution as well as in blood. Previous experimental work provided kinetic data for the catalysis of the 2GSNO → 2NO + GSSG reaction, leading to various proposed mechanisms. Herein, this catalytic process is examined using density functional theory. Minimal functional models of the Cu-MOF cluster and glutathione moieties are established, and three distinct catalytic mechanisms are explored. The most thermodynamically favorable mechanism studied is consistent with prior experimental findings. This mechanism involves coordination of GSNO to copper via sulfur rather than nitrogen and requires a reductive elimination that produces a Cu(I) intermediate, implicating a redox-active copper site. The experimentally observed inhibition of reactivity at high pH values is explained in terms of deprotonation of a triazole linker, which decreases the structural stability of the Cu(I) intermediate. These fundamental mechanistic insights may be generally applicable to other MOF catalysts for NO generation.

Substituent Effects at the N-Nitrosoaminophenol Moiety of a Photoinduced-Electron-Transfer-Driven Nitric Oxide Releaser.

Nitric oxide (NO) has multiple physiological activities, including roles in vasorelaxation, neurotransmission, and immune response. Indeed, NO-releasing compounds are utilized as therapeutic agents for cardiovascular diseases based on the potent and rapid vasorelaxation induced by NO. We have developed a series of photoinduced-electron-transfer-driven (PeT-driven) NO releasers composed of a light-harvesting antenna moiety and an NO-releasing N-nitrosoaminophenol moiety, which efficiently release NO upon irradiation with blue (500 nm), green (560 nm), or red (650 nm) light. In this paper, we investigated substituent effects at the 2-position of the N-nitrosoaminophenol moiety by means of spectroscopic, fluorescence, and NO-release measurements. Interestingly, a methyl substituent at this position had no significant effect on the NO-releasing ability, while a nitro group or a methoxy group reduced it. The nitro group may suppress electron transfer to the antenna moiety, while the methoxy group may accelerate electron transfer but suppress deprotonation to afford the phenoxyl radical, which is the key reaction for release of NO. These structure-activity relationships should be helpful for further functionalizing PeT-driven NO releasers.

Photoinduced NO-release from polymer dots doped with an Ir(III) complex and N-methyl-N-nitroso-4-aminophenol.

Nitric oxide (NO) is a signaling molecule that plays a variety of functions in the human body, but it is difficult to use it in biological experiments or for therapeutic purposes because of its high reactivity and instability in the biological milieu. Consequently, photocontrollable NO releasers, which enable spatiotemporal control of NO release, have an important role in elucidating the functions of NO. Our group has developed visible-light-controllable NO-releasing molecules that contain a fluorescent dye structure as a light-harvesting antenna moiety and an N-nitrosoaminophenol structure as an NO-releasing moiety. Here, we aimed to construct an NO-generating system employing an intermolecular photoredox reaction between the two separate components, since this would simplify chemical synthesis and make it easier to examine various dyes as antennae. For this purpose, we constructed polymer nanoparticles doped with both N-methyl-N-nitroso-4-aminophenol (NAP, 1) and an Ir(III) antenna complex (2, 3 or 4) in order to dissolve in aqueous solution without a co-solvent. These polymer nanoparticles released NO upon photoirradiation in vitro in the purple (400-430 nm) or blue (400-460 nm) wavelength region to activate the doped Ir(III) complex.

Synthesis and Characterization of Nitric Oxide-Releasing Ampicillin as a Potential Strategy for Combatting Bacterial Biofilm Formation.

Biofilm formation on biomaterial interfaces and the development of antibiotic-resistant bacteria have decreased the effectiveness of traditional antibiotic treatment of infections. In this project, ampicillin, a commonly used antibiotic, was conjugated with S-nitroso-N-acetylpenicillamine (SNAP), an S-nitrosothiol compound (RSNO) used for controlled nitric oxide (NO) release. This novel multifunctional molecule is the first of its kind to provide combined antibiotic and NO treatment of infectious pathogens. Characterization of the molecule included NMR, FTIR, and mass spectrometry. NO release behavior was also measured and compared to pure, unmodified SNAP. When evaluating the antimicrobial efficacy, the synthesized SNAPicillin molecule showed the lowest MIC value against Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-resistant Staphylococcus aureus compared to ampicillin and SNAP alone. SNAPicillin also displayed enhanced biofilm dispersal and killing of both bacterial strains when treating a 48 h biofilm preformed on a polymer surface. The antibacterial results combined with the biocompatibility of the molecule show great promise for infection prevention and treatment of polymeric interfaces to reduce medical device-related infections.

A WO3-TiO2 nanorod/CaCO3 photocatalyst with degradation-regeneration double sites for NO2-inhibited and durable photocatalytic NO.

As a promising cleaner technology for nitric oxide degradation, photocatalysis has attracted extensive attention, while the main limitations of photocatalytic nitric oxide are that the toxic NO2 is produced easily and the photocatalytic durability was inferior due to the accumulation of photocatalytic products. In this paper, a WO3-TiO2 nanorod/CaCO3 (TCC) insulating heterojunction photocatalyst with degradation-regeneration double sites was prepared by simple grinding and calcining. The effects of CaCO3 loading on the morphology, microstructure and composition of TCC photocatalyst were investigated by SEM, TEM, XRD, FT-IR and XPS etc. Also, TCC exhibits NO2-inhibited and durable characteristics for NO degradation. DFT calculation, the detection of active radicals by EPR, capture test and the NO degradation pathway characterized by in-situ FT-IR spectra showed that the electron-rich region formed and the existence of regeneration sites are the main reasons for promoting the NO2-inhibited and durable NO degradation. Furthermore, the mechanism of NO2-inhibited and durable NO degradation by TCC was revealed. Finally, TCC superamphiphobic photocatalytic coating was prepared, which still exhibits similar NO2-inhibited and durable characteristics for NO degradation to TCC photocatalyst. It may provide new application value and development prospects in the field of photocatalytic NO.

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.

New nitrosyl ruthenium complexes with combined activities for multiple cardiovascular disorders.

Nitrosyl ruthenium complexes are promising platforms for nitric oxide (NO) and nitroxyl (HNO) release, which exert their therapeutic application. In this context, we developed two polypyridinic compounds with the general formula cis-[Ru(NO)(bpy)2(L)]n+, where L is an imidazole derivative. These species were characterized by spectroscopic and electrochemical techniques, including XANES/EXAFS experiments, and further supported by DFT calculations. Interestingly, assays using selective probes evidenced that both complexes can release HNO on reaction with thiols. This finding was biologically validated by HIF-1α detection. The latter protein is related to angiogenesis and inflammation processes under hypoxic conditions, which is selectively destabilized by nitroxyl. These metal complexes also presented vasodilating properties using isolated rat aorta rings and demonstrated antioxidant properties in free radical scavenging experiments. Based on these results, the new nitrosyl ruthenium compounds showed promising characteristics as potential therapeutic agents for the treatment of cardiovascular conditions such as atherosclerosis, deserving further investigation.