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1.
Eur J Biochem ; 247(2): 688-94, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9266714

ABSTRACT

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mononuclear cupric or met-apo-hemocyanin, and the mononuclear Co(II)-substituted derivative has been studied by near-ultraviolet circular dichroism and EPR spectroscopies. The near-ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper-containing derivatives and a three-component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)-to-metal and to OH -to-metal charge-transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper-containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide-to-Cu(II) charge-transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met-apo-hemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. In the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met-apo-hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal-coordination sphere, identified as a water or hydroxide molecule.


Subject(s)
Azides/metabolism , Cobalt/metabolism , Copper/metabolism , Hemocyanins/chemistry , Hemocyanins/metabolism , Protein Conformation , Animals , Apoproteins/chemistry , Binding Sites , Brachyura , Circular Dichroism , Cobalt/analysis , Copper/analysis , Electron Spin Resonance Spectroscopy , Hemocyanins/isolation & purification
2.
J Inorg Biochem ; 65(3): 167-74, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9025269

ABSTRACT

Copper ions of different types were gradually eliminated from ceruloplasmin (CP1; ferro-O2-oxidoreductase, EC 1.16.3.1.) by dialyzing the enzyme against KCN. Protein was sampled 2, 4, 6, 22, and 28 h after the dialysis started. Atomic absorption allowed us to estimate the amount of copper atoms per CP molecule. Light absorption in the UV and visible regions along with fluorescence and EPR spectra were also registered. Oxidase and dismutase activities of the enzyme were measured at each step. The combination of the data thus obtained allowed us to trace the sequence of CP depletion of certain copper ions. The same methods were applied in reconstitution studies to detect the return of different types of Cu2+. The experiments were performed on CP samples differing in the amount of copper still bound after CN- treatment. It is shown that the oxidase activity is efficiently brought back to CP if, after the dialysis against cyanide, the catalytic center had preserved its type 3 Cu2+. Dismutase activity of CP did not depend greatly on the presence or absence of type 1 and type 2 copper ions. The results obtained allow a more precise evaluation of the role of different types Cu2+ in the assembly of the complex catalytic center of CP and in the accomplishment by the enzyme of its multiple functions.


Subject(s)
Ceruloplasmin/metabolism , Copper/metabolism , Electron Spin Resonance Spectroscopy , Humans , Potassium Cyanide/metabolism , Spectrometry, Fluorescence , Spectrophotometry, Atomic , Spectrophotometry, Ultraviolet
3.
Arch Biochem Biophys ; 313(2): 318-27, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8080279

ABSTRACT

The deoxygenated form of hemocyanin, containing a dinuclear Cu(I) active site, emits luminescence in the red with maximum around 1.54 microns-1 (650 nm). The luminescence of deoxyhemocyanin (deoxy-Hc) from arthropod species is detectable at room temperature, the quantum yield being 2.4-2.7 x 10(-3); in contrast, the emission from molluscan proteins can be detected only at liquid nitrogen temperature. The luminescence emission is an inherent property of the bis[Cu(I)-(histidine)3] complex of the deoxygenated form of the protein to which both Cu(I) ions contribute equally to the overall emission. Luminescence is not observed with the oxygenated and the oxidized forms of hemocyanin, in which the metal is in the Cu(II) state, and in the metal-depleted or apo-Hc form. Based on steady-state and time-resolved measurements and references to Cu(I) model compounds, the luminescence emission is attributed to a triplet excited state of a Cu(I)-to-N (histidine) charge transfer transition 3d-pi*. Acrylamide quenching experiments indicate that the metal active site is very shielded from the solvent. This property of deoxy-Hc enables us to directly follow reactions that modify either the copper oxidation number or the metal-to-protein stoichiometry.


Subject(s)
Calcium/metabolism , Hemocyanins/chemistry , Hemocyanins/metabolism , Animals , Arthropods , Binding Sites , Brachyura , Hydrogen-Ion Concentration , Luminescent Measurements , Mathematics , Mollusca , Octopodiformes , Protein Binding
4.
Biometals ; 7(3): 221-6, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8043987

ABSTRACT

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mM). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.


Subject(s)
Copper/pharmacology , Saccharomyces cerevisiae/drug effects , Adaptation, Physiological , Catalase/physiology , Edetic Acid/pharmacology , Hydrogen-Ion Concentration , Saccharomyces cerevisiae/growth & development , Superoxide Dismutase/physiology
5.
Biophys J ; 65(6): 2680-91, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8312502

ABSTRACT

High-resolution x-ray absorption near edge structure spectroscopy was used to characterize the metal sites in three different cobalt-substituted derivatives of Carcinus maenas hemocyanin (Hc), including a mononuclear cobalt, a dinuclear cobalt and a copper-cobalt hybrid derivative. Co(II) model complexes with structures exemplifying octahedral, trigonal bipyramidal, pseudo-tetrahedral, and square planar geometries were also studied. The results provide structural information about the metal binding site(s) in the Co-Hcs that extend earlier results from EPR and optical spectroscopy (Bubacco et al. 1992. Biochemistry. 31: 9294-9303). Experimental spectra were compared to those calculated for atomic clusters of idealized geometry, generated using a multiple scattering approach. The energy of the dipole forbidden 1s-->3d transition and of the absorption edge in the spectra for all cobalt Hc derivatives confirmed the cobaltous oxidation state which rules out the presence of an oxygenated site. Comparisons between data and simulations showed that the mononuclear and dinuclear Co(II) derivatives, as well as the hybrid derivative, contain four-coordinate Co(II) in distorted tetrahedral sites. Although the spectra for Co(II) in dinuclear metal sites more closely resemble the simulated spectrum for a tetrahedral complex than do spectra for the mononuclear derivative, the Co(II) sites in all derivatives are very similar. The Cu K-edge high resolution x-ray absorption near edge structure spectrum of the hybrid Cu-Co-Hc resembles that of deoxy-Hc demonstrating the presence of three-coordinate Cu(I).


Subject(s)
Cobalt/metabolism , Hemocyanins/chemistry , Metals/analysis , Protein Conformation , Absorptiometry, Photon/methods , Animals , Binding Sites , Brachyura , Cobalt/analysis , Copper/analysis , Electron Spin Resonance Spectroscopy/methods , Hemocyanins/metabolism
6.
Arch Biochem Biophys ; 302(1): 78-84, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8470909

ABSTRACT

The interaction of Carcinus hemocyanin with Cd(II) was studied. The incubation of the apoprotein with the metal yields a derivative containing 1 g-at. of EDTA-stable Cd(II) per 75 kDa. Spectroscopic data ruled out Cd(II) coordination to tryptophan or cysteine residues. The optical activity and fluorescence properties of the protein are affected by Cd(II) binding and indicate a rearrangement of tryptophan residues. The poor Cd(II) binding to the oxy-form and the resistance of Cd(II)-hemocyanin to EDTA treatment and to the regeneration by Cu(I) strongly indicate that Cd(II) binding to apohemocyanin occurs at the copper-free active site. During the metal-binding process, a marked increase of light scattering is observed. This effect, however, is reversible provided that the incubation medium contains SCN- and glycine as exogenous ligands of the metal in the bulk solution.


Subject(s)
Brachyura , Cadmium/metabolism , Hemocyanins/metabolism , Animals , Apoproteins/metabolism , Binding Sites , Cysteine/metabolism , Edetic Acid/pharmacology , Kinetics , Light , Scattering, Radiation , Spectrometry, Fluorescence , Spectrophotometry , Tryptophan/metabolism
7.
Article in English | MEDLINE | ID: mdl-1360381

ABSTRACT

1. Three strains of Saccharomyces cerevisiae have been adapted in vitro upon treatment with copper or cadmium. Growth rate, cellular size, metal uptake, superoxide dismutase and catalase activities were measured. 2. Growth rate and metal uptake are quite different among the yeast strains and also for copper and cadmium treatment. At the employed concentrations, only cadmium chiefly affects the cellular volume. 3. Cu, ZnSOD activity is stimulated in the presence of copper, while it is lightly inhibited in the presence of cadmium. Catalase level remains almost unchanged in the conditions tested. This lack of correlation is then discussed.


Subject(s)
Cadmium/pharmacology , Catalase/metabolism , Copper/pharmacology , Saccharomyces cerevisiae/drug effects , Superoxide Dismutase/metabolism , Cadmium/metabolism , Copper/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development
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