Maria Camilla Baratto

Antioxidant Activity of Galloyl Quinic Derivatives Isolated from P. lentiscus Leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 m ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 w M for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.

EPR characterization of mono(thiosemicarbazones) copper(II) complexes. Note II

Copper(II) complexes with thiosemicarbazones have been shown to be more active in cell destruction, in the inhibition of DNA synthesis than the uncomplexed ligand. Several derivatives of thiosemicarbazones and their iron and copper complexes have been studied for their cytotoxicity and inhibiting activity against DNA synthesis. In the present work complexes formed in H2O-DMSO solution between copper(II) and the acetophenone thiosemicarbazone (ATSC) and the o-aminobenzaldehyde thiosemicarbazone (o-NH2TSC) have been studied. EPR studies have been performed at different pH values and metal-to-ligand ratios. The spectra have been recorded at both room (298 K) and low temperatures (120 K). A possible relationship between structure and activity is attempted on the basis of the EPR data.

Synthesis and reactivity studies on new copper(II) complexes: DNA binding, generation of phenoxyl radical, SOD and nuclease activities

Tridentate ligand PhimpH binds to Cu(II) centre after deprotonation affording a new family of complexes [Cu(Phimp)(H(2)O)](2)(ClO(4))(2) (1), [Cu(Phimp)(2)] (2) and [Cu(Phimp)(L)] (3-5) where L are CH(3)COO(-), SCN(-) and NO(2)(-) respectively. The molecular structures of complexes 1 x CH(3)CN and 3 were determined by X-ray crystallography. Electrochemical studies depicted Cu(II)/Cu(I) couple in the range of -0.50 to -0.65 V vs. Ag/AgCl. EPR spectral studies for complexes 4 and 5 indicated the order of covalent character in 4 > 5 with d(x(2)-y(2)) ground state. Novel copper complexes were synthesized by a tridentate ligand having phenolato, pyridine and azomethine nitrogen donors. Phenoxyl radical complexes were generated; DNA interaction, SOD and nuclease activities were investigated. [corrected]

EPR and O2· — scavenger activity: Cu(II)—peptide complexes as superoxide dismutase models

Several copper(II) complexes with aminoacids and peptides are known to show superoxide dismutase (SOD)-like activity. EPR spectroscopy has proved to be a useful tool for studying the complex equilibria of the copper(II) ion and various ligands of biological importance in solution. In the present work, a variety of copper(II) complexes with di-, tri- and tetra-peptides containing only glycine residues (GG, GGG and GGGG) and others containing a histidyl residue in different positions (HGG, GHG, GGH and GGHG) have been investigated. EPR parameters obtained by extensive use of computer simulation of spectra lead to reliable spin Hamiltonian EPR parameters at both room temperature and in frozen solution. The molecular orbital coefficients computed from the anisotropic EPR data and the d-d electronic energies are used to characterize different arrangements of the complexes. Estimation of the scavenger activity of the complexes due to the particular environment created by the ligands around copper is discussed in the frame of the structure-activity relationship.

Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.

Supramolecular mimetic peroxidase based on hemin and PAMAM dendrimers

An acid-base interaction between hemin and PAMAM dendrimers affords supramolecular non-covalent peroxidase systems whose catalytic activity is enhanced after spontaneous electrostatic self-assembling onto a solid surface.

Prediction model based on decision tree analysis for laccase mediators

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Influence of pH on the speciation of copper(II) in reactions with the green tea polyphenols, epigallocatechin gallate and gallic acid

Changes in speciation of copper(II) in reactions with epigallocatechin gallate (EGCG) and gallic acid (GA) as a function of pH have been investigated by multifrequency (X- and S-band) EPR spectroscopy in the fluid and frozen states. The EPR spectra show the formation of three distinct mononuclear species with each of the polyphenols, and these are interpreted in terms of one mono- and two bis-complexes. However, di- or polymeric complexes dominate the Cu(II) speciation in the pH range 4–8, and it is only at alkaline pH values that these mononuclear complexes make appreciable contributions to the metal speciation. Each mononuclear complex displays linewidth anisotropy in fluid solution as a consequence of incomplete averaging of the spin Hamiltonian parameters through molecular motion. Rotational correlation times for the individual complexes have been estimated by analysing the lineshape anisotropy of the fluid solution spectra using parameters determined by simulation of the rigid limit spectra. These show that the molecular masses increase with increasing pH, indicating either coordination of increasing numbers of polyphenol molecules as ligands to the copper or the increasing involvement of polyphenol dimers as ligands in the copper coordination sphere.

A new program based on stochastic Liouville equation for the analysis of superhyperfine interaction in CW-ESR spectroscopy

In the slow-motion region, ESR spectra cannot be expressed as a sum of simple Lorentzian lines. Studies of Freed and co-workers, on nitroxides in liquids gained information on the microscopic models of rotational dynamics, relying much on computer programs for simulation of ESR spectra based on the stochastic Liouville equation (SLE). However, application of Freeds method to copper system of biological interest has been for a long time precluded by lack of a full program able to simulate ESR spectra containing more than one hyperfine interaction. Direct extension of the Freeds approach in order to include superhyperfine interaction is not difficult from a theoretical point of view but the resulting algorithm is problematical because it leads to substantial increase in the dimensions of the matrix related to the spin-hamiltonian operator. In this paper preliminary results of a new program, written in C, which includes the superhyperfine interactions are presented. This preliminary version of the program does not take into account a restoring potential, so it can be used only in isotropic diffusion conditions. A comparison with an approximate method previously developed in our laboratory, based on a post-convolution approach, is discussed.

Demonstration of lignin-to-peroxidase direct electron transfer. A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR AND NMR STUDY.

Background: Ligninolytic peroxidases are often studied using simple model substrates. Results: Reaction of versatile peroxidase with the lignin polymer is shown by a combination of directed mutagenesis, chromatographic, and spectroscopic techniques. Conclusion: The main reaction implies direct electron transfer to a surface tryptophanyl radical responsible for lignin oxidation. Significance: This is the first time that this central mechanistic aspect is shown for a ligninolytic peroxidase. Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn2+, and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.