Biomedical subjects

Rebecca Pogni

Publications and source records attributed to Rebecca Pogni.

13 recordsLinked to original sources

PubMed2007

Structural and dynamic characterization of copper(II) binding of the human prion protein outside the octarepeat region.

Human prion protein (hPrP) fragments encompassing the 91-120 region, namely hPrP92-100 (SP1), hPrP106-113 (SP2), hPrP91-120 (LP1), and hPrP91-114 (LP2), were considered for delineation of the Cu(II)-binding site(s). NMR and EPR spectroscopy results obtained from LP1 or LP2 were compared with those obtained from SP1 and SP2. The coexistence of two binding sites, one centered at His96 and the other at His111, was evidenced and ratified by ESI mass spectrometry at low and high metal:peptide ratios. While room-temperature NMR spectroscopy data were consistent with the binding site centered on His111 being approximately fourfold stronger than that centered on His96, low-temperature EPR spectroscopy results yielded evidence for the opposite trend. This disagreement, which has also occurred in the literature, was clarified by temperature-dependent molecular dynamics runs that demonstrated Met112 approaching the metal at room temperature, a process that is expected to stabilize the His111-centered binding site through hydrophobic shielding of the metal coordination sphere.

Binding Sites↗

PubMed2006

Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering.

Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.

Amino Acid Sequence↗

PubMed2006

Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: a SDSL-EPR and MD study.

Five singly spin labeled side chains at surface sites in the C-terminal domain of RGL2 protein have been analyzed to investigate the general relationship between nitroxide side chain mobility and protein structure. At these sites, the structural perturbation produced by replacement of a native residue with a nitroxide side chain appears to be very slight at the level of the backbone fold. The primary determinants of the nitroxide side chain mobility are backbone dynamics and tertiary interactions. On the exposed surfaces of alpha-helices, the side chain mobility is not restricted by tertiary interactions but appears to be determined by backbone dynamics, while in loop sites, the side chain mobility is even higher. For a better understanding of the changes in the EPR spectral line shape, molecular dynamics simulations were performed and found in agreement with EPR spectral data.

Electron Spin Resonance Spectroscopy↗

PubMed2006

Mechanism of versatile peroxidase inactivation by Ca(2+) depletion.

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.

Basidiomycota↗

PubMed2006

A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multifrequency EPR and density functional theory study.

Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn(2+) as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H(2)O(2) has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp(164) as the site involved in long-range electron transfer for aromatic substrate oxidation.

Catalysis↗

PubMed2005

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways.

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.

Catalysis↗

PubMed2005

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta.

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.

Basidiomycota↗

PubMed2005

Interaction of the human prion PrP(106-126) sequence with copper(II), manganese(II), and zinc(II): NMR and EPR studies.

The synthetic peptide encompassing residues 106-126 (PrP106-126, KTNMKHMAGAAAAGAVVGGLG) of the human prion protein was considered for its binding properties toward copper(II), manganese(II) and zinc(II) at pH 5.7. 1H and 13C 1D spectra, 1H spin-lattice relaxation rates, and 1H-15N and 1H-13C HSQC 2D experiments were obtained in the absence and in the presence of metal ions. While Zn(II) was found to yield negligible effects upon any NMR parameter, metal-peptide association was demonstrated by the paramagnetic effects of Cu(II) and Mn(II) upon 1D and 2D spectra. Delineation of structures of metal complexes was sought by interpreting the paramagnetic effect on 1H spin-lattice relaxation rates. Exchange of peptide molecules from the metal coordination sphere was shown to provide sizable contribution to the observed relaxation rates. Such contribution was calculated in the case of Cu(II); whereas the faster paramagnetic rates of peptide molecules bound to Mn(II) were determining spin-lattice relaxation rates almost exclusively dominated by exchange. Proton-metal distances were therefore evaluated in the case of the Cu(II) complex only and used as restraints in molecular dynamics calculations where from the structure of the complex was obtained. The peptide was shown to bind copper through the imidazole nitrogen and the ionized amide nitrogen of His-111 and the amino-terminal group with the terminal carboxyl stabilizing the coordination sphere through ionic interactions. The data were interpreted as to demonstrate that the hydrophobic C-terminal region was not affecting the copper-binding properties of the peptide and that this hydrophobic tail is left free to interact with other target molecules. As for the complex with Mn(II), qualitative information was obtained on carbonyl oxygens of Gly-124 and Leu-125, beyond the terminal Gly-126 carboxyl, being at close distance from the metal ion, that also interacts, most likely, through a hydrogen bond of metal-bound water, with the imidazole ring of His-111.

Amino Acid Sequence↗

PubMed2004

Electron paramagnetic resonance studies on copper(II)-cyclodextrin systems.

Three copper(II) complexes containing beta-cyclodextrin (betaCD) derivatives as ligands [mono-6-methylamino-6-deoxy-betaCD dithiocarbamate (CDTC), mono-6-histamino-6-deoxy-betaCD (CDHM) and mono-6-Nalpha-arginyl-6-deoxy-betaCD (CDARG)] have been studied by electron paramagnetic resonance. The spectra have been recorded at X- and S-bands and different temperatures and simulated to obtain the best set of magnetic parameters. In particular, the 300 K spectra are typical of the slow motion regime, as expected considering the high molecular weight of the ligands. Some structural characteristics of the complexes are proposed on the basis of dynamic and covalency parameters obtained from simulation.

Copper↗

PubMed2003

Synthesis and molecular modelling studies of resorcin[4]arene-capped porphyrins.

Three new resorcin[4]arene-capped porphyrins (3, 5 and 7) different in the porphyrin skeleton, in the linking arms and in the cavity dimensions, have been synthesised. Molecular modelling calculations explored the conformations and the cavity size of the three compounds and showed that their hydrophobic pockets can accommodate one molecule of water or methane (3 and 5), or benzene (7) without any distortion. Notably, the capped porphyrin 5 was able to inhibit the oxidation of Co(II) to Co(III), whereas compound 7 did it only partially.

Journal Article↗

PubMed2003

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 Freed's 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 Freed's 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.

Anisotropy↗

PubMed2003

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 (O2) 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 (IC50) of di- and tri-galloyl derivatives did not exceed 30 microM 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.

Antioxidants↗

PubMed2002

Synthesis and interaction with copper(II) cations of cyano- and aminoresorcin[4]arenes.

Following our studies on resorcin[4]arenes, we synthesized new macrocycles containing cyanomethyl and aminomethyl side chains. Three stereoisomers (2a-c) of the former were obtained by BF3*Et2O tetramerization of the corresponding trans-cinnamic acid derivative and were shown to be in the 1,2-alternate, cone, and 1,3-alternate conformations. Conversely, the tetraamino derivative 6a in the cone conformation was prepared from the corresponding tetrabromide 3a. The interactions with Cu(II) cations of the new compounds were analyzed by measurements of 1H NMR and EPR spectra in parallel with molecular modeling calculations.

Journal Article↗