Copper(II)-Assisted Degradation of Pheophytin a by Reactive Oxygen Species.

The central ion Mg2+ is responsible for the differences between chlorophyll a and its free base in their reactivity toward metal ions and thus their resistance to oxidation. We present here the results of spectroscopic (electronic absorption and emission, circular dichroism, and electron paramagnetic resonance), spectroelectrochemical, and computational (based on density functional theory) investigations into the mechanism of pheophytin, a degradation that occurs in the presence of Cu ions and O2. The processes leading to the formation of the linear form of tetrapyrrole are very complex and involve the weakening of the methine bridge due to an electron withdrawal by Cu(II) and the activation of O2, which provides protection to the free ends of the opening macrocycle. These mechanistic insights are related to the naturally occurring damage to the photosynthetic apparatus of plants growing on metal-contaminated soils.

A Personal Account on Inorganic Reaction Mechanisms.

The presented Review is focused on the latest research in the field of inorganic chemistry performed by the van Eldik group and his collaborators. The first part of the manuscript concentrates on the interaction of nitric oxide and its derivatives with biologically important compounds. We summarized mechanistic information on the interaction between model porphyrin systems (microperoxidase) and NO as well as the recent studies on the formation of nitrosylcobalamin (CblNO). The following sections cover the characterization of the Ru(II)/Ru(III) mixed-valence ion-pair complexes, including Ru(II)/Ru(III)(edta) complexes. The last part concerns the latest mechanistic information on the DFT techniques applications. Each section presents the most important results with the mechanistic interpretations.

Investigation of the Immunogenic Properties of Ovalbumin Modified by Urban Airborne Particulate Matter.

Exposure to air particulate matter (PM) is linked to the blood oxidative stress and systemic inflammation. The aim of this study was to elucidate whether oxidative PM modification of ovalbumin (OVA), the major antioxidant serum protein, may alter its antigenicity and/or immunogenicity. Ovalbumin was exposed via dialysis to the standard urban PM (SRM 1648a) or to PM with removed organic content (encoded as LAP). Both structural changes and biological properties of PM-modified OVA were measured. T lymphocytes and dendritic cells (the major antigen-presenting cells) isolated from C57BL/6 and OT-II (323-339 epitope) OVA-specific T cell receptor (TCR)-transgenic mice were used to test the effect of PM on OVA immunogenicity. The immunogenicity of both SRM 1648a and LAP-modified OVA was significantly higher than that of control OVA, as measured by the epitope-specific T cell proliferation and interferon γ production by the stimulated cells. This effect was associated with mild oxidative changes in the carrier molecule outside the structure of the OVA epitope and with increased resistance to proteolysis of PM-modified OVA. Interestingly, dendritic cells showed enhanced capacity for the uptake of proteins when the cells were cultured with PM-modified OVA. Our results suggest that the enhanced immunogenicity of PM-modified OVA is not associated with altered antigenicity or antigen presentation. However, it may result from slower degradation and longer persistence of modified antigens in dendritic cells. Whether this phenomenon is associated with enhanced risk prevalence of autoimmune diseases observed in the areas with high urban PM pollution needs to be explained.

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.

Relevance of the electron transfer pathway in photodynamic activity of Ru(II) polypyridyl complexes containing 4,7-diphenyl-1,10-phenanthroline ligands under normoxic and hypoxic conditions.

The purpose of this study was to investigate the correlation between the spectroscopic and photophysical properties of Ru(II) polypyridyl complexes and their photodynamic activity in vitro. A series of Ru(II) polypyridyl complexes with 4,7-diphenyl-1,10-phenanthroline (dip) and 2,3-bis(2-pyridyl)quinoxaline (dpq) and its derivatives were synthesized and characterized regarding their photophysical, biological, and photodynamic properties. The complexes were evaluated not only in the context of 1O2 generation but also regarding other types of reactive oxygen species (ROS) to assess the possibility of Ru(II) complexes to induce phototoxicity via various ROS using fluorescence and EPR spectroscopy. The compounds were found to be moderately cytotoxic with IC50 values ranging from 1 to 35 µM and retained their cytotoxic activity under hypoxic conditions. The unraveled phototoxic activity is based mainly on the generation of H2O2 and 1O2, highlighting the importance of electron-transfer processes in the observed photodynamic activity of Ru polypyridyl complexes. A combination of photodynamic activity with cytotoxicity under decreased dioxygen concentrations may help overcome the current photodynamic therapy (PDT) limitation. The findings highlight the need for broadening the scope of tested Ru-based photosensitizers.

Evaluation of anticancer activity in vitro of a stable copper(I) complex with phosphine-peptide conjugate.

[CuI(2,9-dimethyl-1,10-phenanthroline)P(p-OCH3-Ph)2CH2SarcosineGlycine] (1-MPSG), highly stable in physiological media phosphino copper(I) complex-is proposed herein as a viable alternative to anticancer platinum-based drugs. It is noteworthy that, 1-MPSG significantly and selectively reduced cell viability in a 3D spheroidal model of human lung adenocarcinoma (A549), in comparison with non-cancerous HaCaT cells. Confocal microscopy and an ICP-MS analysis showed that 1-MPSG effectively accumulates inside A549 cells with colocalization in mitochondria and nuclei. A precise cytometric analysis revealed a predominance of apoptosis over the other types of cell death. In the case of HaCaT cells, the overall cytotoxicity was significantly lower, indicating the selective activity of 1-MPSG towards cancer cells. Apoptosis also manifested itself in a decrease in mitochondrial membrane potential along with the activation of caspases-3/9. Moreover, the caspase inhibitor (Z-VAD-FMK) pretreatment led to decreased level of apoptosis (more pronouncedly in A549 cells than in non-cancerous HaCaT cells) and further validated the caspases dependence in 1-MPSG-induced apoptosis. Furthermore, the 1-MPSG complex presumably induces the changes in the cell cycle leading to G2/M phase arrest in a dose-dependent manner. It was also observed that the 1-MPSG mediated intracellular ROS alterations in A549 and HaCaT cells. These results, proved by fluorescence spectroscopy, and flow cytometry, suggest that investigated Cu(I) compound may trigger apoptosis also through ROS generation.

Blood Plasma's Protective Ability against the Degradation of S-Nitrosoglutathione under the Influence of Air-Pollution-Derived Metal Ions in Patients with Exacerbation of Heart Failure and Coronary Artery Disease.

One of the consequences of long-term exposure to air pollutants is increased mortality and deterioration of life parameters, especially among people diagnosed with cardiovascular diseases (CVD) or impaired respiratory system. Aqueous soluble inorganic components of airborne particulate matter containing redox-active transition metal ions affect the stability of S-nitrosothiols and disrupt the balance in the homeostasis of nitric oxide. Blood plasma's protective ability against the decomposition of S-nitrosoglutathione (GSNO) under the influence of aqueous PM extract among patients with exacerbation of heart failure and coronary artery disease was studied and compared with a group of healthy volunteers. In the environment of CVD patients' plasma, NO release from GSNO was facilitated compared to the plasma of healthy controls, and the addition of ascorbic acid boosted this process. Model studies with albumin revealed that the amount of free thiol groups is one of the crucial factors in GSNO decomposition. The correlation between the concentration of NO released and -SH level in blood plasma supports this conclusion. Complementary studies on gamma-glutamyltranspeptidase activity and ICP-MS multielement analysis of CVD patients' plasma samples in comparison to a healthy control group provide broader insights into the mechanism of cardiovascular risk development induced by air pollution.

Experimental and Computational Insight into the Mechanism of NO Binding to Ferric Microperoxidase. The Likely Role of Tautomerization to Account for the pH Dependence.

According to the current paradigm, the metal-hydroxo bond in a six-coordinate porphyrin complex is believed to be significantly less reactive in ligand substitution than the analogous metal-aqua bond, due to a much higher strength of the former bond. Here, we report kinetic studies for nitric oxide (NO) binding to a heme-protein model, acetylated microperoxidase-11 (AcMP-11), that challenge this paradigm. In the studied pH range 7.4-12.6, ferric AcMP-11 exists in three acid-base forms, assigned in the literature as [(AcMP-11)FeIII(H2O)(HisH)] (1), [(AcMP-11)FeIII(OH)(HisH)] (2), and [(AcMP-11)FeIII(OH)(His-)] (3). From the pH dependence of the second-order rate constant for NO binding (kon), we determined individual rate constants characterizing forms 1-3, revealing only a ca. 10-fold decrease in the NO binding rate on going from 1 (kon(1) = 3.8 × 106 M-1 s-1) to 2 (kon(2) = 4.0 × 105 M-1 s-1) and the inertness of 3. These findings lead to the abandonment of the dissociatively activated mechanism, in which the reaction rate can be directly correlated with the Fe-OH bond energy, as the mechanistic explanation for the process with regard to 2. The reactivity of 2 is accounted for through the existence of a tautomeric equilibrium between the major [(AcMP-11)FeIII(OH)(HisH)] (2a) and minor [(AcMP-11)FeIII(H2O)(His-)] (2b) species, of which the second one is assigned as the NO binding target due to its labile Fe-OH2 bond. The proposed mechanism is further substantiated by quantum-chemical calculations, which confirmed both the significant labilization of the Fe-OH2 bond in the [(AcMP-11)FeIII(H2O)(His-)] tautomer and the feasibility of the tautomer formation, especially after introducing empirical corrections to the computed relative acidities of the H2O and HisH ligands based on the experimental pKa values. It is shown that the "effective lability" of the axial ligand (OH-/H2O) in 2 may be comparable to the lability of the H2O ligand in 1.

Ligand-Tuning of the Stability of Pd(II) Conjugates with Cyanocobalamin.

Besides the well-known functions performed by vitamin B12 (CblCN) in biochemical processes of the human body, an increasing interest has been raised by the possibility of its use as a transmembrane drug carrier, capable, among others, of enhancing the accumulation of inorganic cytostatics in cancer cells. The present study was aimed at determining the possibility of the formation of CblCN conjugates with Pd(II) complexes. A key aspect was their stability, which we attempted to tune by appropriate choice of ligands. Syntheses, spectroscopic analysis of postreaction systems and kinetic investigations of conjugate formation reactions, have been complemented by DFT modelling. The obtained results showed that ligand charge, geometry and electron affinity may have a significant impact on carrier binding and release leading to the activation of the Pd(II) complex. This provides a rationale to expect that with appropriate composition of the coordination sphere, it will be possible to extend the spectrum of less toxic inorganic chemotherapeutics.

High-Pressure Mechanistic Insight into Bioinorganic NO Chemistry.

Pressure is one of the most important parameters controlling the kinetics of chemical reactions. The ability to combine high-pressure techniques with time-resolved spectroscopy has provided a powerful tool in the study of reaction mechanisms. This review is focused on the supporting role of high-pressure kinetic and spectroscopic methods in the exploration of nitric oxide bioinorganic chemistry. Nitric oxide and other reactive nitrogen species (RNS) are important biological mediators involved in both physiological and pathological processes. Understanding molecular mechanisms of their interactions with redox-active metal/non-metal centers in biological targets, such as cofactors, prosthetic groups, and proteins, is crucial for the improved therapy of various diseases. The present review is an attempt to demonstrate how the application of high-pressure kinetic and spectroscopic methods can add additional information, thus enabling the mechanistic interpretation of various NO bioinorganic reactions.

Enhancement of NO release from S-nitrosoalbumin by pollution derived metal ions.

S-Nitrosothiols act as a comparatively long-lived reservoir of releasable nitric oxide (NO) present in vivo in a variety of body fluids. Soluble constituents of air-borne particulate matter (PM) can affect S-nitrosothiol stability and deregulate NO-based biological signaling. PM aqueous extracts of standard urban dust (SRM 1648a) were prepared, and their effect on human serum S-nitrosoalbumin (HSA-NO) stability was studied. The results indicated that PM extracts induced a release of NO from HSA-NO in a dose-dependent manner. To identify the inorganic components of urban PM responsible for HSA-NO decomposition, the effects of individual metal ions and metal ion mixtures, detected in the SRM 1648a aqueous extract, were examined. The dominant role of copper ions (specifically Cu+) was confirmed, but the results did not exclude the influence of other water-soluble PM components. Measurements with the application of several common metal ion chelators confirmed that Cu2+ may participate in NO release from HSA-NO and that reduction to monovalent Cu+ (responsible for S-NO bond breaking) may occur with the participation of S-nitrosoalbumin. The addition of ascorbic acid (AscA) significantly enhanced the effectiveness of NO release by PM extracts both kinetically and quantitatively, by inducing an increase in the reduction of Cu2+ to Cu+. These results indicate that AscA present in the respiratory tract lining fluids and plasma may amplify the activity of inorganic components of PM in S-nitrosothiol decomposition.

Mechanistic Studies on the Reaction between Aquacobalamin and the HNO Donor Piloty's Acid over a Wide pH Range in Aqueous Solution.

Detailed kinetic and mechanistic studies have been carried out on the reaction between aquacobalamin/hydroxocobalamin (CblOH2+/CblOH) and nitroxyl (HNO) generated by Piloty's acid (PA, N-hydroxybenzenesulfonamide) over a wide pH range (3.5-13). The resulting data showed that in a basic solution HNO can react with hydroxocobalamin to form nitrosylcobalamin despite the inert nature of CblOH. It was shown that at low PA concentrations the rate-determining step is the decomposition of PhSO2NHO- to release HNO, whereas the reaction between CblOH and HNO becomes the rate-determining step at high PA concentrations. Data from kinetic studies on the reaction of CblOH with an excess of HNO enabled us to experimentally determine the pKa(HNO) value from initial rate data as a function of pH, giving pKa(HNO) = 11.47 ± 0.04. An especially interesting observation was made in the neutral pH range, where PA is stable and does not produce HNO. Under such conditions, rapid formation of CblNO was observed in the studied system. The obtained data suggest that CblOH2+ reacts directly with PA to form a Piloty's acid-bound cobalamin intermediate, which deprotonates rapidly at neutral pH followed by rate-determining S-N bond cleavage to give CblNO and release PhSO2-.

Chlorophyll a π-Cation Radical as Redox Mediator in Superoxide Dismutase (SOD) Mimetics.

The extensive speciation of copper(II) chloride in organic solvents varies with concentration, temperature, pressure and oxygen content, providing the ability to switch between different chlorophyll transmetalation pathways. We found that one of them is exceptionally suitable for the formation and stabilisation of the chlorophyll π-cation radical. This is due to unique redox cycling, which is coupled to the generation and transformation of various reactive oxygen species. In the presence of a proton donor, our system shows behavior which resembles that of superoxide dismutase (SOD). Regardless of light, chlorophyll acts as an electron transfer mediator.

Enhanced Cellular Uptake and Photodynamic Effect with Amphiphilic Fluorinated Porphyrins: The Role of Sulfoester Groups and the Nature of Reactive Oxygen Species.

A class of amphiphilic photosensitizers for photodynamic therapy (PDT) was developed. Sulfonate esters of modified porphyrins bearing-F substituents in the ortho positions of the phenyl rings have adequate properties for PDT, including absorption in the red, increased cellular uptake, favorable intracellular localization, low cytotoxicity, and high phototoxicity against A549 (human lung adenocarcinoma) and CT26 (murine colon carcinoma) cells. Moreover, the role of type I and type II photochemical processes was assessed by fluorescent probes specific for various reactive oxygen species (ROS). The photodynamic effect is improved not only by enhanced cellular uptake but also by the high generation of both singlet oxygen and oxygen-centered radicals. All of the presented results support the idea that the rational design of photosensitizers for PDT can be further improved by better understanding the determinants affecting its therapeutic efficiency and explain how smart structural modifications can make them suitable photosensitizers for application in PDT.

Variations in Reactive Oxygen Species Generation by Urban Airborne Particulate Matter in Lung Epithelial Cells-Impact of Inorganic Fraction.

Air pollution is associated with numerous negative effects on human health. The toxicity of organic components of air pollution is well-recognized, while the impact of their inorganic counterparts in the overall toxicity is still a matter of various discussions. The influence of airborne particulate matter (PM) and their inorganic components on biological function of human alveolar-like epithelial cells (A549) was investigated in vitro. A novel treatment protocol based on covering culture plates with PM allowed increasing the studied pollutant concentrations and prolonging their incubation time without cell exposure on physical suffocation and mechanical disturbance. PM decreased the viability of A549 cells and disrupted their mitochondrial membrane potential and calcium homeostasis. For the first time, the difference in the reactive oxygen species (ROS) profiles generated by organic and inorganic counterparts of PM was shown. Singlet oxygen generation was observed only after treatment of cells with inorganic fraction of PM, while hydrogen peroxide, hydroxyl radical, and superoxide anion radical were induced after exposure of A549 cells to both PM and their inorganic fraction.

Nitrosyl- versus nitroxyl-cobalamin?

The Commentary is in answer to the comment of a reader that objected against the use of the term 'nitroxylcobalamin' in two recent reports in JBC from our group. We use this opportunity to explain to the reader where this terminology originated from.

Urban Particulate Matter-Induced Decomposition of S-Nitrosoglutathione Relevant to Aberrant Nitric Oxide Biological Signaling.

Exposure to airborne particulate matter (PM) is associated with hazardous effects on human health. Soluble constituents of PM may be released in biological fluids and disturb the precisely tuned nitric oxide signaling processes. The influence of aqueous extracts from two types of airborne urban PM (SRM 1648a, a commercially available sample, and KR PM2.5, a sample collected "in-house" in Krakow, Poland) on the stability of S-nitrosoglutathione (GSNO) was investigated. The particle interfaces had no direct effect on the studied reaction, but extracts obtained from both samples facilitated NO release from GSNO. The effectiveness of NO release was significantly affected by glutathione (GSH) and ascorbic acid (AscA). Examination of the combined influence of Cu2+ , Fe3+ , and reductants on GSNO stability revealed copper to be the main GSNO decomposing species. Computational models of nitrosothiols interacting with metal oxide substrates and solvated metal ions support these claims. The study stresses the importance of the interplay between metal ions and biological reductants in S-nitrosothiols decomposition.

Generation of hydroxyl radicals and singlet oxygen by particulate matter and its inorganic components.

Particulate matter (PM) can strongly affect redox biochemistry and therefore induce the response of the immune system and aggravate the course of autoimmune diseases. Nanoparticles containing transition metal compounds possessing semiconductor properties (TiO2, ZnO) may act as photocatalysts and accelerate the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In this study, the NIST standard reference material, SRM 1648a, has been analyzed in terms of this consideration. Organic compounds present in SRM 1648a were removed by cold oxygen plasma treatment. Samples of SRM 1648a with removed organic content (<2% of organic carbon, <1% of nitrogen) were obtained within 2 h of this treatment. The treatment did not affect the morphology of the powder. The reference material and PM2.5 collected in Kraków are composed of smaller particles and nanoparticles forming aggregates. The efficiency of (photo)generation of hydroxyl radicals and singlet oxygen was compared for original and organics-free samples. The analyzed samples showed the highest activity towards ROS generation when exposed to UV-vis-NIR light, moderate under UV irradiation, and the lowest in dark. Data collected in the present study suggest that the organic fraction is mostly responsible for singlet oxygen generation, as almost twice higher efficiency of 1O2 generation was observed for the original NIST sample compared to the material without the organic fraction. However, particulate matter collected in Kraków was found to have a five times higher activity in singlet oxygen generation (compared for original NIST and Kraków dust samples).

Can nitrocobalamin be reduced by ascorbic acid to nitroxylcobalamin? Some surprising mechanistic findings.

Despite detailed studies on nitroxylcobalamin (CblNO) formation, the possible intracellular generation of CblNO via reduction of nitrocobalamin (CblNO2) remains questionable. To study this further, spectroscopic studies on the reaction of CblNO2 with the intracellular antioxidant ascorbic acid (HAsc-) were performed in aqueous solution at pH < 5.0. It was found that nitroxylcobalamin is the final product of this interaction, which is not just a simple reaction but a rather complex chemical process. We clearly show that an excess of nitrite suppresses the formation of CblNO, from which it follows that ascorbic acid cannot reduce coordinated nitrite. We propose that under the influence of ascorbic acid, nitrocobalamin is reduced to Cbl(II) and nitric oxide (·NO), which can subsequently react rapidly to form CblNO. It was further shown that this system requires anaerobic conditions as a result of the rapid oxidation of both Cbl(II) and CblNO.

Activation volumes for cis-to-trans isomerisation reactions of azophenols: a clear mechanistic indicator?

The thermal cis-to-trans isomerisation reaction of a series of hydroxy-substituted azo derivatives was studied kinetico-mechanistically as a function of temperature and pressure in order to investigate the possible role of the solvent in controlling the isomerisation mechanism, viz. inversion versus rotation. The variation of the observed first order rate constants for kinetic runs carried out at different temperatures and pressures was used to determine the thermal activation parameters ΔH‡ and ΔS‡, and the pressure activation parameter ΔV‡. In addition, some experiments with deuterated species or solvents were also performed. The reported results could be interpreted as indicative of a changeover from an inversion mechanism for non-polar solvents to a rotational mechanism for polar solvents, capable of hydrogen bonding, for some of the systems studied. However, the operation of a rotational mechanism in all studied cases can account more consistently for the data observed.