Methionine-121 coordination determines metal specificity in unfolded<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> azurin

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.     

Removal of copper from Octopus vulgaris haemocyanin. Preparation of the half-apo and apo derivatives.

The two copper ions bound in the active site of Octopus vulgaris haemocyanin can be removed by cyanide. The two metal ions react with the ligand sequentially. In this paper the preparation of Octopus half-apo-haemocyanin, containing at the active site a single copper ion, is described. Moreover, the conditions to obtain Octopus apo-haemocyanin, containing less than 3% of copper still bound, are given.     

Interaction of copper(II) and nickel(II) with a bis-amide ligand functionalized with pyridine moieties: Thermodynamic and spectroscopic studies in aqueous solution

We synthesized a new bis-amide ligand derived from the l(+)-tartaric acid. We then determined its protonation constants and the stability constants of the copper(II) and nickel(II) chelates by potentiometry as well as ESI-MS and UV-Vis spectroscopy. We found that both metal ions are able to induce the deprotonation and the coordination of an amide nitrogen donor atom. In the case of copper complexes, the data show the formation of two major species: Cu₂(L₂H₋₃)⁺ and Cu₂(LH₋₄). EPR and XAS experiments led us to precise the relative structure of these compounds. In Cu₂(L₂H₋₃)⁺, each metal center is coordinated by pyridinic and amidic nitrogen atoms of one ligand and by nitrogen and oxygen atoms from pyridine and hydroxyl moieties from the other one. In Cu₂(LH₋₄), the copper centers are coordinated by pyridinic and amidic nitrogen atoms, as well as a deprotonated hydroxyl group of the ligand. In this latter complex, the lower value of the Cu-Cu distance determined from EXAFS experiments and compared to the one of the solid species likely involve the formation of an exogeneous hydroxyl bridge between the two copper centers. With Ni(II) ions, the only one major species is the mononuclear Ni(LH₋₂) complex, in which Ni(II) is held in an octahedral environment with the metal center chelated by the two pyridinic and the two amidic nitrogen atoms, and two oxygen atoms from water molecules.     

Synthesis, characterisation, X-ray structure and biological activity of three new 5-formyluracil thiosemicarbazone complexes

Three new complexes of transition metals as copper, nickel and cobalt with 5-formyluracil thiosemicarbazone (H3ut) have been synthesised and characterised by single-crystal X-ray diffraction. In all compounds the ligand behaves as SNO terdentate. In the copper complex the coordination geometry is square pyramidal with the ligand lying on the basal plane and two water molecules that complete the metal environment, the nickel compound is surrounded by six donor atoms (three of the ligand, two water oxygen atoms and a chlorine atom) in an octahedral fashion, and cobalt also shows an octahedral geometry but determined only by two terdentate ligand molecules. These three compounds have been tested on human leukemic cell lines K562 and CEM. The nickel and cobalt complexes have demonstrated low activity in cell growth, while the copper complex that is more active has been tested also on a third leukemic human cell line (U937), but it was not able to induce apoptosis on all cell lines.     

Metal binding characteristics of human salivary and porcine pancreatic amylase

With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.     

The biotic ligand model and a cellular approach to class B metal aquatic toxicity

The biotic ligand model (BLM) and a cellular molecular mechanism approach represent two approaches to the correlation of metal speciation with observed toxicity to aquatic organisms. The two approaches are examined in some detail with particular reference to class B, or soft metals. Kinetic arguments are presented to suggest situations that can arise where the BLM criterion of equilibrium between all metal species in the bulk solution and the biotic ligand may not be satisfied and what might the consequences be to BLM predictive capability. Molecular mechanisms of toxicity are discussed in terms of how a class B metal might enter a cell, how it is distributed in a cell, and how the cell might respond to the unwanted metal. Specific examples are given for copper as an organism trace essential metal, which is toxic in excess, and for silver, a non-essential metal. As class B metals all bind strongly to sulfur, regulation of these metals requires that all S(II-) species be accounted for in aquatic systems, even under oxic conditions.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

Crystallographic and NMR investigation of cobalt-substituted amicyanin

Cobalt(II) amicyanin was prepared by replacing the copper of the type I copper protein amicyanin from Paracoccus denitrificans with cobalt. The structure of the protein and the metal center have been characterized by X-ray crystallography and paramagnetic NMR spectroscopy. The crystal structure indicates that Met98, which provides an axial sulfur ligand in native amicyanin, is no longer bound to the metal in cobalt(II) amicyanin and that a water molecule is recruited from solvent to form the fourth metal ligand. This results in a tetrahedral coordination geometry for the cobalt ion. NMR studies in solution also indicate that the side chain of the methionine residue interacts less strongly with the metal in P. denitrificans amicyanin than in Paracoccus versutus amicyanin. The cobalt(II) amicyanin crystal structure is different from that of cobalt-substituted azurin in which the carbonyl of a glycine residue provides this equivalent ligand. In cobalt(II) amicyanin that residue is a proline, for which the oxygen is structurally inaccessible, so that the water occupies the position held by the glycine carbonyl in cobalt(II) azurin. Such a metal coordination involving water has not previously been reported for a native or metal-substituted type I copper protein.     

Synthesis, antimicrobial activity and chemotherapeutic potential of inorganic derivatives of 2-(4'-thiazolyl)benzimidazole[thiabendazole]: X-ray crystal structures of [Cu(TBZH)2Cl]Cl.H2O.EtOH and TBZH2NO3 (TBZH=thiabendazole)

Thiabendazole (TBZH) reacts with iron(III) nitrate causing protonation of the ligand to yield the nitrate salt [TBZH(2)NO(3)] (1). Reaction of TBZH with copper(II) acetate results in the deprotonation of the ligand yielding [Cu(TBZ)2.(H2O)2] (2). Reactions of TBZH with the chloride, nitrate and butanedioate salts of copper(II) yields [Cu(TBZH)2Cl]Cl.H2O.EtOH (3), [Cu(TBZH)(2)(NO(3))(2)] (4) and [Cu(TBZH)(O(2)C-CH(2)CH(2)-CO(2))] (5), respectively. The TBZH acts as a neutral chelating ligand in 3-5. Molecular structures of 1 and 3 were determined crystallographically. In 1, the asymmetric unit contains one TBZH(2)(+) cation and one NO(3)(-) anion. The structure of 3 comprises a five coordinate copper centre with the metal bound to two chelating TBZH ligands and one chloride. The geometry is best described as trigonal bipyramidal. Hydrogen bonding connects the complex cation with the uncoordinated chloride anion and the water and ethanol solvate molecules. Compound 1 and the copper complexes 2-5, the metal free ligands and a number of simple copper(II) salts were each tested for their ability to inhibit the growth of Candida albicans. The metal free TBZH and its nitrate salt (1) exhibited very poor activity. Complex 2, in which the TBZH is present as an anionic ligand (TBZ(-)), exhibits moderate activity towards the pathogen. Chelation of the neutral TBZH to copper centres (complexes 3-5) results in potent anti-candida activity. The dimethyl sulphoxide (DMSO) soluble complexes 3 and 4, along with metal free TBZH were assessed for their cancer chemotherapeutic potential towards two human epithelial-derived cancer model cell lines. Complexes 3 and 4 displayed similar dose-dependent cytotoxicity in both cell lines with IC(50) values of approximately 50 microM, which were found to be significantly lower than that for metal free TBZH.     

Copper complex-assisted DNA hybridization

2,2'-Bipyridine (bpy) or 1,10-phenanthroline (phen) metal-binding domains were covalently attached to oligonucleotides, and the influence of metal ions on the hybridization of the conjugates was investigated. Metal-binding domains were attached to oligonucleotides at 3'- and 5'-terminal positions, thus placing them in juxtaposed positions after hybridization to a common target strand. While the ligands alone had a positive effect (increased Tm) on hybrid stability, the duplex was further stabilized by the addition of copper(I) and/or copper(II) through the formation of a metal complex in which the two short sequences are linked through {Cu(bpy)2}, {Cu(phen)}, or {Cu(bpy)(phen)} domains. The increase in Tm, due to formation of the {Cu(bpy)2}, {Cu(phen)2}, {Cu(bpy)(phen)} motifs is reversed upon addition of EDTA, consistent with the stripping of copper from the ligands. The effect of metal complex formation on the duplex strength was shown to be highest if the two metal-coordinating ligand strands are placed as close to each other as possible.     

A ligand field model for MCD spectra of biological cupric complexes.

A ligand field calculation of magnetic circular dichroism (MCD) spectra is described that provides new insights into the information contained in electronic spectra of copper sites in metalloenzymes and synthetic analogs. The ligand field model uses metal-centered p- and f-orbitals to model sigma, pi LMCT mixing mechanism for intensity, allowing the basic features of optical absorption, MCD, and electron paramagnetic resonance spectra to be simultaneously computed from a single set of parameters and the crystallographically determined ligand coordinates. We have used the model to predict changes in spectra resulting from the transformation of electronic wavefunctions under systematic variation in geometry in pentacoordinate ML5 complexes. The effectiveness of the calculation is demonstrated for two synthetic copper model compounds and a galactose oxidase enzyme complex representing limiting coordination geometries. This analysis permits immediate recognition of characteristic patterns of MCD intensity and correlation with geometry. A complementarity principle between MCD and CD spectra of transition metal complexes is discussed.     

Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (相似文献   

Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA,two essential components of Escherichia coli pco copper resistance operon

The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway. Periplasmic extracts show copper-inducible oxidase activity, attributed to the multicopper oxidase function of PcoA. PcoC, a much smaller protein than PcoA, binds one Cu(II) and exhibits a weak electronic transition characteristic of a type II copper center. ENDOR and ESEEM spectroscopy of Cu(II)-PcoC and the (15)N- and Met-CD(3)-labeled samples are consistent with a tetragonal ligand environment of three nitrogens and one aqua ligand "in the plane". A weakly associated S-Met and aqua are likely axial ligands. At least one N is a histidine and is likely trans to the in-plane aqua ligand. The copper chemistry of PcoC and the oxidase function of PcoA are consistent with the emerging picture of the chromosomally encoded copper homeostasis apparatus in the E. coli cell envelope [Outten, F. W., Huffman, D. L., Hale, J. A., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 30670-30677]. We propose a model for the plasmid system in which Cu(I)-PcoC functions in this copper efflux pathway as a periplasmic copper binding protein that docks with the multiple repeats of Met-rich domains in PcoA to effect oxidation of Cu(I) to the less toxic Cu(II) form. The solvent accessibility of the Cu(II) in PcoC may allow for metal transfer to other plasmid and chromosomal factors and thus facilitate removal of Cu(II) from the cell envelope.     

Probing copper ligands in denatured <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> azurin: unfolding His117Gly and His46Gly mutants

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.     

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a beta-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D(2)O/H(2)O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short alpha-helix) and the last beta-strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.     

Properties of the binding of copper by bleomycin

The glycopeptide, bleomycin, binds metal ions including Cu²⁺. It is the copper complex of this material that is isolated from Streptomyces verticillus. Both free ligand and copper complex are excellent antitumor agents in animals. The biochemical and pharmacological relationship between these compounds has not been established. The present study begins an analysis of the chemistry and biochemistry of copper-bleomycin with structural and equilibrium properties of the complex. Potentiometric and fluorometric titrations of bleomycin confirm three acidic groups with pK_a values of 7.50, 4.93, and 2.72. The conjugate nitrogen bases of these groups, comprise three of the binding sites for Cu²⁺ according to similar titrations of copper-bleomycin. The fourth is a conjugate base of an acid with a very large pK_a that cannot be measured by these techniques. The participation of a fourth such group is inferred from both proton release studies of the binding of metal and ligand above pH 8 and from several studies of the thermodynamic stability of copper bleomycin. At low pH binding of copper to bleomycin occurs in two steps, as observed by several independent techniques which monitor either the metal or the ligand. Log stability constants for the reactions Cu²⁺ + H_kBlm ? CuH_k-nBlm + nH⁺ and CuH_k-nBlm ? CuH_k-n-rBlm + rH⁺ are 1.32 and ?4.31, respectively, with n of 2.21 in the first equation and r of 2.07 in the second equation. The derived logarithm of the pH independent stability constant for copper bleomycin multiplied by the protonation constant for the unknown fourth ligand in the binding site is 12.16. This agrees closely with values obtained from measurements of conditional formation constants. One of the groups which binds in the second reaction is the substituted pyrimidine.     

Solution structure of Apo Cu,Zn superoxide dismutase: role of metal ions in protein folding

The solution structure of the demetalated copper, zinc superoxide dismutase is obtained for the monomeric Glu133Gln/Phe50Glu/Gly51Glu mutant through NMR spectroscopy. The demetalated protein still has a well-defined tertiary structure; however, two beta-strands containing two copper ligands (His46 and His48, beta4) and one zinc ligand (Asp83, beta5) are shortened, and the sheet formed by these strands and strands beta7 and beta8 moves away from the other strands of the beta-barrel to form an open clam with respect to a closed conformation in the holoprotein. Furthermore, loop IV which contains three zinc ligands (His63, His71, and His80) and loop VII which contributes to the definition of the active cavity channel are severely disordered, and experience extensive mobility as it results from thorough (15)N relaxation measurements. These structural and mobility data, if compared with those of the copper-depleted protein and holoprotein, point out the role of each metal ion in the protein folding, leading to the final tertiary structure of the holoprotein, and provide hints for the mechanisms of metal delivery by metal chaperones.     

Speciation of copper(II) in natural waters in the presence of ligands of high and intermediate strength

Abstract

A new procedure is presented for the determination of the ligands of copper(II) in natural waters, based on titration with the metal ion, monitored by measuring the concentration of copper(II) sorbed on the carboxylic resin Amberlite CG 50. The data are treated by the Ruzic linearization method to obtain the concentration of the ligands and the conditional stability constant of the complexes. Ligands with reaction coefficient α_M higher than 0.1 K*w/V are detected, where K* is the ratio of the concentration of sorbed metal to the concentration of free metal in solution, which can be evaluated from the sorption equilibria of copper(II) on Amberlite CG 50, w is the amount of water in the resin phase, and V the volume of the solution phase. Some natural waters at high and low salinity were examined. The ligand concentration determined in these samples ranged from around 50 to 2000 nM, while the original copper concentrations from 11 to 130 nM. The ligand concentration was always much higher than that of copper(II). The conditional stability constants were very high, particularly in low salinity waters, where values as high as K’= 10^15.7 were obtained. In high salinity waters values around 10⁹ were found for the complex formation constant of the ligands titrated with copper(II). The investigation was also extended to a model solution, containing EDTA, obtaining K’ = 10^15.5, in acceptable agreement with that evaluated from the literature values.     

Synthesis and characterization of metal ion imprinted nano-porous polymer for the selective recognition of copper

Selective recognition of metal ions utilizing metal ion-imprinted polymers (MIIPs) received much importance in diverse fields owing to their high selectivity for the target metal ions. In the present study, a copper ion imprinted polymer was synthesized without an additional complexing ligand or complex with a broad aim to avoid the conventional extra metal ion complexing ligand during the synthesis of MIIP. The complete removal of the copper metal ion from the MIIP was confirmed by AAS and SEM–EDX. SEM image of the MIIP exhibited nano-patterns and it was also found to be entirely different from that of non-imprinted polymer and polymer with copper metal ions. BET surface area analysis revealed more surface area (47.96 m²/g) for the Cu(II)-MIIP than non-imprinted control polymer (41.43 m²/g). TGA result of polymer with copper metal ion indicated more char yield (18.41%) when compared to non-imprinted control polymer (8.3%) and Cu(II)-MIIP (less than 1%). FTIR study confirmed the complexation between Cu(II)-MIIP and Cu(II) metal ion through carbonyl oxygen of acryl amide. The Cu(II)-MIIP exhibited an imprinting efficiency of 2.0 and it was showing 8% interference from a mixture of Zn, Ni and Co ions. A potentiometric ion selective electrode devised with Cu(II)-MIIP showed more potential response for Cu(II) ion than that was fabricated from non-imprinted polymer.     

Functionally specified protein signatures distinctive for each of the different blue copper proteins

Background  

Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands.