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The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.

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The reactivity of nitrite towards the copper(II) and copper(I) centers of a series of complexes with tridentate nitrogen donor ligands has been investigated. The ligands are bis[(1-methylbenzimidazol-2-yl)methyl]amine (1-bb), bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine (2-bb), and bis[2-(3,5-dimethyl-1-pyrazolyl)ethyl]amine (ddah) and carry two terminal benzimidazole (1-bb, 2-bb) or pyrazole (ddah) rings and a central amine donor residue. While 2-bb and ddah form two adjacent six-membered chelate rings on metal coordination, 1-bb forms two smaller rings of five members. The binding affinity of nitrite and azide to the Cu(II) complexes (ClO4- as counterion) has been determined in solution. The association constants for the two ligands are similar, but nitrite is a slightly stronger ligand than azide when it binds as a bidentate donor. The X-ray crystal structure of the nitrite complex [Cu(ddah)(NO2)]ClO4 (final R=0.056) has been determined: triclinic P1space group, a=8.200(2) A, b=9.582(3) A, c=15.541(4) A. It may be described as a perchlorate salt of a "supramolecular" species resulting from the assembly of two complex cations and one sodium perchlorate unit. The copper stereochemistry in the complex is intermediate between SPY and TBP, and nitrite binds to Cu(II) asymmetrically, with Cu-O distances of 2.037(2) and 2.390(3) A and a nearly planar CuO2N cycle. On standing, solutions of [Cu(ddah)(NO2)]ClO4 in methanol produce the dinuclear complex [Cu(ddah)(OMe)]2(ClO4)2, containing dibridging methoxy groups. In fact the crystal structure analysis (final R=0.083) showed that the crystals are built up by dinuclear cations, arranged on a crystallographic symmetry center, and perchlorate anions. Electrochemical analysis shows that binding of nitrite to the Cu(II) complexes of 2-bb and ddah shifts the reduction potential of the Cu(II)/Cu(I) couple towards negative values by about 0.3 V. The thermodynamic parameters of the Cu(II)/Cu(I) electron transfer have also been analyzed. The mechanism of reductive activation of nitrite to nitric oxide by the Cu(I) complexes of 1-bb, 2-bb, and ddah has been studied. The reaction requires two protons per molecule of nitrite and Cu(I). Kinetic experiments show that the reaction is first order in [Cu(I)] and [H+] and exhibits saturation behavior with respect to nitrite concentration. The kinetic data show that [Cu(2-bb)]+ is more efficient than [Cu(1-bb)]+ and [Cu(ddah)]+ in reducing nitrite.

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The complex [Cu2(L-66)]2+ (L-66 = a,a'-bis¿bis[2-(1'-methyl-2'-benzimidazolyl)ethyl]amino¿-m-xylene) undergoes fully reversible oxygenation at low temperature in acetone. The optical [lambda(max) = 362 (epsilon 15000), 455 (epsilon 2000), and 550 nm (epsilon 900M(-1)cm(-1))] and resonance Raman features (760 cm(-1), shifted to 719cm(-1)(-1) with 18O2) of the dioxygen adduct [Cu2(L-66)(O2)]2+ indicate that it is a mu-eta2:eta2-peroxodicopper(II) complex. The kinetics of dioxygen binding, studied at - 78 degrees C, gave the rate constant k1 = 1.1M(-1) 5(-1) for adduct formation, and k(-1) =7.8 x 10(-5)s(-1), for dioxygen release from the Cu2O2 complex. From these values, the O2 binding constant K= 1.4 x 10(4)M(-1) at -78 degrees C could be determined. The [Cu2(L-66)(O2)]2+ complex performs the regiospecific ortho-hydroxylation of 4-carbomethoxyphenolate to the corresponding catecholate and the oxidation of 3,5-di-tert-butylcatechol to the quinone at -60 degrees C. Therefore, [Cu2(L-66)]2+ is the first synthetic complex to form a stable dioxygen adduct and exhibit true tyrosinase-like activity on exogenous phenolic compounds.

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Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by ascorbate oxidase was investigated at pH 7.0 and 23.0 degrees C. Inhibition of p-nitrophenol is pH dependent over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1 copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol bound to ascorbate oxidase are between 4.4 and 5.9 A.

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The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.

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Bleeding from small bowel is a quite rare event and often is a diagnostic challenge to physician and surgeon. We present a case of a patient with an acute massive haemorrhage due to jejunal diverticulosis and with an unusual clinical setting. The site of bleeding was localized by scan with radiotagged erythrocytes, but the diagnosis of jejunal diverticule was evident only at laparotomy. The patient underwent to surgical resection of the affected bowel (40 cm). Although jejunal diverticula are considered a rare source of gastrointestinal haemorrhage, we suggest that this disorder must be considered in all patients with occult gastrointestinal bleeding especially in the elderly.

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The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050-1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43-108, J. Wiley, New York]. Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and preceds the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M-1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M-1), where a second azide ligand also binds in the terminal mode (K = 20 M-1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.

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The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity.

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The binding of a series of alkyl aryl sulfides to chloroperoxidase (CPO) and horseradish peroxidase (HRP) has been investigated by optical difference spectroscopy, circular dichroism, paramagnetic NMR spectroscopy, and NMR relaxation measurements. The data are consistent with binding of the sulfides in the distal side of the heme pocket with CPO and near the heme edge with HRP. A linear correlation between the binding constants of para-substituted sulfides to CPO and the Taft sigma I parameter suggests that these substrates act as donors in donor-acceptor complexes involving some residue of the protein chain. Spectral studies during turnover show that high enantioselectivity in the CPO-catalyzed oxidation of sulfides results from a reaction pathway that does not involve the accumulation of compound II enzyme intermediate.

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The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.

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The interaction of a series of derivatives of tyrosine with horseradish peroxidase (HRP) and lactoperoxidase (LPO) was studied by using optical difference spectroscopy, c.d. and proton n.m.r. spectroscopy in order to reveal differences in the mode of binding of L-tyrosine and D-tyrosine, which are substrates of but react at different rates with the two peroxidases, to HRP and LPO. All the donor molecules form 1:1 complexes with HRP and LPO, but they display a range of affinities for the enzymes. Whereas D-tyrosine binds to HRP more strongly than does L-tyrosine, the opposite holds for the binding to LPO. The distances of the protons of bound tyrosine molecules from the haem iron atoms of HRP and LPO indicate that the site of binding of these substrates is the same as that of simple phenols. This involves the interaction of the phenol nucleus with a protein tyrosine residue [Sakurada, Takahashi & Hosoya (1986) J. Biol. Chem. 261, 9657-9662; Modi, Behere & Mitra (1989) Biochim. Biophys. Acta 996, 214-225]. However, for the present substrates the additional interaction of the carboxylate group with a protein residue (probably an arginine residue) provides further stabilization for the adducts HRP-D-tyrosine and LPO-L-tyrosine with respect to the corresponding complexes with the opposite enantiomers. The differences in the mode of binding of L-tyrosine and D-tyrosine to HRP and LPO is thus determined by the fact that the spatial arrangement of the interacting protein residues can recognize the chirality of the C(alpha)-CO2- and C(beta)-C6H4OH attachment bonds of the substrates.

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The chloroperoxidase-catalyzed and horseradish peroxidase catalyzed oxidations of sulfides by tert-butyl and other peroxides have been investigated. The former metal enzyme afforded the corresponding sulfoxides having R absolute configuration in up to 92% enantiomeric excess (ee), whereas the latter gave racemic products. The various factors that control the enantioselectivity of the oxygenation have been examined.

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A case of spontaneous gallbladder-cutaneous fistula is reported with emphasis on the rarity of this lesion after the introduction of cholecystectomy.

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Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.

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The coordination structures of various species in the copper(II)-L-histidine (1:2) system in aqueous solution have been deduced by investigating the pH dependence of the electronic and circular dichroism spectra. The contribution to the spectra of the glycine-like and histamine-like binding modes of L-histidine has been determined by recording the spectra of the ternary system copper(II)-histamine-L-histidine (1:1:1) and copper(II)-amino acid-L-histidine (1:1:1), respectively, in neutral aqueous solutions. Apical binding to copper(II) by the donor atom on the histidine side chain can contribute significantly to the stabilization of each of the two basic histidine binding modes. It has been concluded that Cu(HL)2+ (L-histidine = HL), the major species below pH approximately 3, contains a glycine-like bound histidine ligand with an unbound imidazolium cation. The species Cu(HL)L+, which is prominent in the pH region near 4.5, contains a glycine-like bound histidine molecule, with protonated imidazole ring, and a histamine-like bound histidine molecule. CuL2, the major species at neutral pH, exists in solution as an equilibrium mixture of a mixed-type chelation structure, with a glycine-like and a histamine-like bound histidine ligand, and a structure containing both histidine ligands bound histamine-like. The species containing deprotonated imidazole nuclei, such as Cu(H-1L2)-, which predominates above pH approximately 11, show an increased contribution by structures containing glycine-like bound histidine compared with CuL2.

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