Tiziana Beringhelli

Hemoglobin and heme scavenging

Release of hemoglobin into plasma is a physiological phenomenon associated with intravascular hemolysis. In plasma, stable haptoglobin‐hemoglobin complexes are formed and these are subsequently delivered to the reticulo‐endothelial system by CD163 receptor‐mediated endocytosis. Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma. Albumin, haptoglobin, hemopexin, and high and low density lipoproteins cooperate to trap the plasma heme, thereby ensuring its complete clearance. Then hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin‐heme complex by CD91 receptor‐mediated endocytosis. Moreover, α1‐microglobulin contributes to heme degradation by a still unknown mechanism, with the concomitant formation of heterogeneous yellow‐brown kynurenine‐derived chromophores which are very tightly bound to amino acid residues close to the rim of the lipocalin pocket. During hemoglobin synthesis, the erythroid α‐chain hemoglobin‐stabilizing protein specifically binds free α‐hemoglobin subunits limiting the free protein toxicity. Although highly toxic because capable of catalyzing free radical formation, heme is also a major and readily available source of iron for pathogenic organisms. Gram‐negative bacteria pick up the heme‐bound iron through the secretion of a hemophore that takes up either free heme or heme bound to heme‐proteins and transports it to a specific receptor, which, in turn, releases the heme and hence iron into the bacterium. Here, hemoglobin and heme trapping mechanisms are summarized. IUBMB Life, 57: 749‐759, 2005

Oxidation reaction of [{Cu(Hpz)2Cl}2](Hpz = pyrazole): synthesis of the trinuclear copper(II) hydroxo complexes [Cu3(OH)(pz)3(Hpz)2Cl2]·solv (solv = H2O or tetrahydrofuran). Formation, magnetic properties, and X-ray crystal structure of [Cu3(OH)(pz)3(py)2Cl2]·py (py = pyridine)

The trinuclear copper(II) complexes [Cu3(OH)(pz)3(Hpz)2Cl2]·solv (Hpz = pyrazole, pz = pyrazolate anion, solv = H2O or tetrahydrofuran) have been obtained by oxidation reactions of [{Cu(Hpz)2Cl}2]. When treated with pyridine(py), both CuII derivatives gave the related compound [Cu3(OH)(pz)3-(py)2Cl2]·py, which has been characterized by a single-crystal X-ray structure analysis. Crystals are orthorhombic, space group Pnma(no. 62), a= 19.883(3), b= 15.063(3), c= 9.495(2)A, Z= 4, R= 0.035 and R′= 0.041 for 1 472 absorption corrected reflections having I > 3σ(I).

Reorganization in apo- and holo-β-lactoglobulin upon protonation of Glu89: Molecular dynamics and PKa calculations

Molecular dynamics (MD) simulations starting from crystallographic data allowed us to directly account for the effects of the protonation state of Glu89 on the conformational stability of apo‐ and holo‐β‐lactoglobulin (BLG). In apo‐BLG simulations starting from the protonated crystal structure, we observe a long‐lived H‐bond interaction between the protonated Glu89 and Ser116. This interaction, sequestering the proton from the aqueous medium, explains a pKhalf value evaluated at pH 7.3 by continuum electrostatics/Monte Carlo computation on MD data, using linear response approximation. A very large root‐mean‐square deviation (RMSD; 5.11 Å) is observed for the EF loop between protonated and unprotonated apo‐BLG. This results from a quite different orientation of the EF loop that acts either as a closed or as an open lid above the protein calyx. Proton exchange by Glu89 in apo‐ but not in holo‐BLG is associated with a reorganization energy of 4.7 kcal/mol. A 3‐ns MD simulation starting from the crystal structure of protonated apo‐BLG, but considering the Glu89 as unprotonated, shows the progressive opening of the lid giving rise to the Tanford transition. In both holo‐BLG forms, the lid is most probably held in place by hydrophobic interactions of amino acid side‐chains of the EF loop with the palmitate hydrocarbon tail. Proteins 2004;00:000–000.

Spectroscopic and spectromagnetic study of adenosine chloroderivatives of bivalent Co, Ni, Cu, Zn, Cd. Comparison with the corresponding adenine chloroderivatives

Abstract [M″LnCl2] (n = 1,2) (M″= Co, Ni, Cu, Zn, Cd, Hg) (L = adenine, adenosine) compounds were synthesized and the magnetic, electronic and vibrational properties of adenosine derivatives were considered in comparison with those of adenine complexes. It was found that the ligand field symmetry is distorted tetrahedral in cobalt, zinc compounds and in CuLCl2; distorted octahedral in Ni, Cd, Hg compounds and in CuL2Cl2. Sugar moiety of adenosine is not implied in the coordination to the metal center. Distortions from regular symmetries were recognized by ESR measurements and confirmed by vibrational analysis; it resulted an higher distorting power of adenosine than of adenine. Both monodentate and bridging N(3)—N(9) or N(1)—N(7) coordination were discussed. The MN bond strength resulted higher for bridged coordination. ESR sensitive change of spin state were observed in CoL2Cl2 compounds. Vibrational measurements on purine absorption modes suggest that the ligand is not strongly perturbed by the metal coordination.

Bovine β-lactoglobulin acts as an acid-resistant drug carrier by exploiting its diverse binding regions

Abstract Binding of fluorine-containing drugs to bovine β-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of 19F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where β-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine β-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine β-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.

Rhenium(V) oxide complexes. Crystal and molecular structures of ReOI2(PPh3)2(OReO3) and of ReOI2(PPh3)(OPPh3)(OReO3)·0.5C6H6 obtained from the reaction of ReO2I(PPh3)2 with oxygen

The oxygenation reaction of the five-coordinated violet species ReO 2 I(PPh 3 ) 2 leads to isolation of two crystalline compounds, both characterized by X-ray diffraction analysis: the dark-red ReOI 2 (PPh 3 ) 2 (OReO 3 ) (1), which is the main product, and the yellow ReOI 2 (PPh 3 )(OPPh 3 )(OReO 3 )·0.5C 6 H 6 (2). Compound 1 gives monoclinic crystals, space group C 2/ c , with a = 25.033(8), b = 16.944(5), c = 19.495(8) A, β = 116.13(3)°, Z = 8. Compound 2 is monoclinic, space group P 1 / n , with a = 9.498(4), b = 25.048(8), c = 17.148(5) A, β = 92.47(3)°, Z = 4. The structures of 1 and 2 were solved by Patterson and Fourier methods and refined by full-matrix least-squares, on the basis of 4229 ( 1 ) and 2672 ( 2 ) significant counter data ( I > 3σ( I )), respectively. The final values of the conventional agreement indices R and R w were 0.050 and 0.070 ( 1 ) and 0.033 and 0.039 (2), respectively. Both species contain a previously unexpected coordinated perrhenato ligand. The geometry of the two complexes is distorted octahedral: compound 1 contains two trans phosphine ligands (mean ReP= 2.513 A) and two trans iodides (mean ReI 2.730 A), the other two trans coordination sites being occupied by the oxide (ReO = 1.670(7) A) and the perrhenate. Compound 2 shows a similar coordination geometry, with one of the phosphine ligands replaced by a phosphine oxide. The main bond parameters in 2 are: ReP 2.453(4) A, ReI (mean) 2.727 A, ReO(OPPh 3 ) 2.075(9) A, ReO (oxide) 1.639(9) A. The interactions Re(V)-perrhenate are similar in the two complexes, with Re(V)O bonds of 2.031(6) A ( 1 ) and 2.079(9) A ( 2 ). The ReORe angles show some difference: 164.3(4)° ( 1 ) and 153.5(5)° ( 2 ). Within the perrhenates, the ReO bond involving the bridging oxygen atom is somewhat longer than the other ReO bonds.

An Intramolecular NH⋅⋅⋅(-H)Re2 Dihydrogen Bond and a Novel 3-2 Coordination Mode of the Pyrazolate Anion on a Triangular Cluster Face

The quantitative addition of pyrazole (Hpz) to the 44 valence-electron, triangular cluster anion [Re3(μ3-H)(μ-H)3(CO)9]− gives the novel unsaturated anion [Re3(μ-H)4(CO)9(Hpz)]− (1, 46 valence electrons), which contains a pyrazole molecule that is terminally coordinated on a cluster vertex. Solid-state X-ray and IR analyses reveal a rather weak hydrogen-bonding interaction between the NH proton and one of the hydrides bridging the opposite triangular cluster edge (ΔH°=−3.1 kcal mol−1 from the Iogansen equation). Both IR and NMR data indicate that such a proton–hydride interaction is maintained in the major conformer present in CD2Cl2, but also provide evidence of the presence of minor conformers of 1 in which the NH proton is involved in an intermolecular hydrogen bond with the solvent. The μ-H⋅⋅⋅HN bond length evaluated in solution through the T1 minimum value (2.07 A) and that determined in the solid state by X-ray diffraction (2.05 A) are in good agreement. NMR experiments show that, in acetone, intermolecular NH⋅⋅⋅solvent interactions replace the intramolecular dihydrogen bond. At room temperature in CH2Cl2, the pyrazole ligand in 1 is labile and 1 slowly “disproportionates” to [Re3(μ3-H)(μ-H)3(CO)9]− and [Re3(μ-H)3(CO)9(μ-η2-pz)(Hpz)]−, with H2 evolution. Slow H2 evolution also leads to the formation of the anion [Re3(μ-H)3(CO)9(pz)]− (5), in which the pyrazolate anion adopts a novel μ3-η2-coordination mode, as revealed by a single-crystal X-ray analysis. The analysis of the bond lengths indicates that the pyrazolate anion in 5 acts as a six-electron donor, with loss of the aromaticity. The formation of 5 from 1 is much faster in solvents with a high dielectric constant, such as acetone or DMF. Anion 5 was also obtained from the reaction of pyrazole with [Re3(μ-H)3(CO)9(μ3-CH3)]− through the intermediate formation of two isomeric addition derivatives and following CH4 evolution.

Synthesis and characterization of copper(I)-pyrazole complexes. X-Ray crystal structure of [Cu(pzH)2Cl]2 and NMR investigation of the fluxional behaviour of [Cu(pzH)2(CO)Cl]

Abstract The product of the reduction of [Cu(pzH) 4 Cl 2 ] (pzH = pyrazole, C 3 H 4 N 2 ) with Cu powder has been shown by single crystal X-ray analysis to the dimeric species [Cu(pzH) 2 Cl] 2 . The crystals are monoclinic, space group P 2 1 / n , with a 6.327(1), b 9.610(1), c 15.066(1) A, β 96.91(1), and Z = 2. The refinements performed by full-matrix least-squares methods, for 1417 independent significant reflexions, gave a final R value of 0.030. Each dimeric unit, located on a crystallographic inversion centre, contains two Cu atoms (Cu…Cu′ 3.401(1) A), each surrounded by two bridging C1 atoms and two pzH ligands, in a pseudo-tetrahedral arrangement. The reaction of carbon monoxide with [Cu(pzH) 2 Cl] 2 gave the new species [Cu(pzH) 2 (CO)Cl], which was characterized by IR and NMR spectroscopy. Fluxional behaviour of the pyrazole ligands, equalizing their 3 and 5 positions, was revealed by both 1 H and 13 C NMR spectra. Variable temperature 13 C NMR experiments in different conditions (solvent or concentrations) lead to different E a values, indicating that the exchange process is not intramolecular, and is favoured in donor solvents, at temperatures above 250 K. In concentrated solutions in CD 2 Cl 2 a double-slope Arrhenius plot was obtained, suggesting the occurrence of two different processes, whose natures are discussed.

ESR investigation of paramagnetic carbonyl-metal clusters of high nuclearity

Abstract An ESR study has been made of the high nuclearity paramagnetic metal cluster anions [Rh12(CO)13(μ2-CO)10(C)2]3-, [Co13(CO)12(μ2-CO)12(C)2]4- and [Co6(CO)8(μ2-CO)6C]-. The assignment of the HOMO is based on a mixed valence model which relates the g tensor components of cluster systems to those of an appropriate conventional paramagnetic center. With this model the HOMOs of [Rh12(CO)13(μ2-CO)10(C)2]3- and of [Co13(CO)12(μ2-CO)12(C)2]4- are found to be mainly comprised of metal dz2 atomic orbitals, while for [Co6(CO)8(μ2-CO)6C]- a large overlap between d atomic orbitals and ligand orbitals is suggested. The occupation of the valence molecular orbitals deduced from the ESR data is consistent with the variations in MM bond distance observed by X-ray analysis.

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL‐BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL‐BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo‐ and holo‐cL‐BABP. Most water molecules identified in the binding site of the apo‐crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo‐crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein‐ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL‐BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL‐BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo‐ and holo‐cL‐BABP but are lost immediately after the start of molecular dynamics. Copyright