Cu2+ probe of metal-ion binding sites in melanin using electron paramagentic resonance spectroscopy. II. Natural melanin

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of natural melanin from the choroid of bovine eyes using electron paramagnetic spectroscopy. Samples were in aqueous media over a wide range of pH values. At pH < 7, binding is to monodentate carboxyl complexes and to bidentate nitrogen-carboxyl complexes just as in synthetic melanin. At pH > 7 binding is to phenolic hydroxyl groups, but the number of such sites is much less than in synthetic melanin and there are indications of a superimposed spectrum of another site. At high pH, a signal unlike any found in synthetic melanin was observed with either three or four nitrogen ligands. A number of experiments indicate that natural melanin is 50% protein by weight. Metal-ion binding sites are the same with and without protein although with some differences in relative populations.     

Ultrastructural and chemical distinction of melanins formed by Verticillium dahliae from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine

Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed malanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenlalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but no catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH⁻ substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Binding of metal ions to polysaccharides. VI. Spectroscopic and potentiometric studies of the binding of copper(II) and nickel(II) to some monosaccharide units in acidic,neutral and alkaline aqueous media

Binding of Cu²⁺ and Ni²⁺ to glucosamine, N-acetyl- glucosamine and other derivatives of glucose was investigated in acidic, neutral and alkaline aqueous media using H⁺ and Cu²⁺ potentiometry and ligand- field and ESR spectroscopy. In neutral medium, site binding with copper(II) and nickel(II) occurs when the monosaccharide possesses a potentially coordinating amine or charged group not attached to C-1. At high pH, a coordination entity is only formed if the C-1 hydroxyl group can be deprotonated and other stabilizing groups are present. The role of groups attached to C-1 reflects the different behaviour of monosaccharides compared with polysaccharides.     

Catechol Oxidase and SOD Mimicking by Copper(II) Complexes of Multihistidine Peptides

Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis–Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis–Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {N_im/2N_im,2N^?} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range.     

Metal chelates in the storage and transport of neurotransmitters: interactions of Cu2+ with ATP and biogenic amines

Abstract— The thermodynamic stabilities of the coordinate binding of Cu²⁺ ion with adenosinetriphos-phate (ATP) and several biogenic amines have been determined in aqueous model systems in an attempt to examine the possible correlation between metal-amine binding and the in vivo affinities of the amines for granule-binding. In each of the ternary chelate systems consisting of Cu²⁺-ATP-amine (1:1:1), the Cu²⁺ ion is preferentially bound by ATP in the pH range 3–5 with a stability constant of Log K_ML= 517. In the pH range 5–8 each of the biogenic amines coordinates with Cu²⁺ -ATP chelate to form the respective ternary chelate. The nature and strength of binding of fourteen different amines with Cu²⁺-ATP have been evaluated on the basis of the stabilities of the ternary chelates. On the basis of the quantitative equilibrium data generated in this study, it appears that both pyrocatechol moiety and the ethanolamine side-chain of the catechol amines are involved in the coordination of copper. The metal-binding stabilities of the biogenic amines are then correlated with the molecular structure, donor basicities and the in vivo affinities of the amines for granule-binding in order to rationalize the possible involvement of metal chelates in the monoamine binding, storage and transport.     

The configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrP^C) is a Cu²⁺ binding protein connected to the outer cell membrane. The molecular features of the Cu²⁺ binding sites have been investigated and characterized by spectroscopic experiments on PrP^C-derived peptides and the recombinant human full-length PrP^C (hPrP-[23-231]). The hPrP-[23-231] was loaded with ⁶³Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP^C/Cu²⁺-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu²⁺ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu²⁺ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP^C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electron paramagnetic resonance evidence of metal-ion binding sites in Micrococcus lysodeikticus (M. luteus) F1-ATPase

Evidence is presented for the presence of divalent cation binding sites in purified F₁-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F₁-ATPase indicate that the enzyme binds Mn²⁺ and Cu²⁺. Scatchard-type plot for Mn²⁺ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10⁻⁶ M. Cu²⁺ binds to the enzyme at only one kind of site(s). This Cu²⁺ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg²⁺ binds at these Mn²⁺ and Cu²⁺ binding sites.     

Thermodynamics and kinetics of the interaction of copper (II) ions with native DNA.

Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu²⁺ ions with native DNA at low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation at low relative ligand concentrations and at low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu²⁺ ions with atoms N₍₇₎ of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases, as well as the formation of specific intestrand crosslink complexes at adjacent G°C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA–Cu²⁺)_nat may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs at increased copper concentrations and/or at increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA–Cu²⁺)_denat, indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA. From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu²⁺ ions to atoms N₍₃₎ of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G·C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu²⁺ ions between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G·C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu²⁺, Cd²⁺, and Zn²⁺ ions with DNA. In temperature-jump experiments with the nondenatured complex (DNA–Cu²⁺)_nat, a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perturbation. The resulting sigmoidal shape of the kinetic curves is considered to be a consequence of the necessity of disrupting a certain number of the crosslinks existing in the nondenatured complex before the local unwinding of the binding regions (a main step of denaturing complex formation) may proceed.     

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.     

Mn 2+ , and Zn 2+ 1:1 complexes with biochemically significant thioether carboxylic acids and some of the sulfoxide and sulfone derivatives

The 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with S-carboxymethyl alkyl and S-carboxymethyl aryl mercaptans were studied in water containing 50% dioxane (I = 0.1; t = 25 °). The determination of the stability constants and a comparison with simple carboxylate complexes reveals that the complexes of Cu²⁺ (and slightly also of Zn²⁺) with the S-carboxymethyl alkyl mercaptans are more stable than expected from only basicity of the carboxylate groups. This suggests that the thioether group participates in complex formation, i.e., chelates are formed. The Mn²⁺ complexes of both kinds of ligands, and the Cu²⁺ or Zn²⁺ complexes with S-carboxymethyl aryl mercaptans have the stability expected according to the basicity of the carboxylate groups. NMR experiments with S-carboxymethyl ethyl mercaptan confirm the formation of chelates with Cu²⁺ and suggest simple carboxylate complexes with Mn²⁺. Analogous experiments with (S-carboxymethyl phenyl mercaptan do not allow an unequivocal statement about the distribution between simple carboxylate complexes and chelates for both metal ions. Also, as the thioether acids are biologically oxidized, the complex stabilities of several of such oxidized derivatives were measured.     

Interaction of Aplysia brasiliana Myoglobin with Mn2+ and Cu2+ ions

Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu²⁺ and Mn²⁺ with MbApB. In the present work Cu²⁺ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH₃ lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu²⁺ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant K_a = ( 11.5 ± 0.8) 10³M⁻¹. The binding of Cu²⁺ to MbApB is stronger than Mn²⁺, K_a^Cu2+ >K_a^Mn2+.     

Analytical determination of apparent stability constants using a copper ion selective electrode

Copper(II) complexes of di-, tri- and tetra peptides with previously published protonation constants were re-investigated using pH and copper ion selective electrode (ISE) potentiometry in conjunction with a modified version of HYPERQUAD computer program. The purpose was to demonstrate the suitability of the ISE approach for the determination of apparent stability constants for copper(II) complexes with ligands for which proton stability constants were not available. The interactions of Cu²⁺ with oligopeptides were also analysed using surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-ToF-MS). The results provide an insight into the metal complex species formed, their apparent stabilities under selected conditions and the effect of the relative positions of certain amino acids within the peptide sequence.     

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu²⁺ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail.MethodsWe investigated the residue specific mapping of Cu²⁺ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu²⁺ were studied using fluorescence, CD, and AFM respectively.ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu²⁺ showed an enhanced rate of fibril formation with a pronounced effect for G51D.ConclusionCu²⁺ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu²⁺ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q.General SignificanceThese findings will help in the better understanding of Cu²⁺ catalyzed aggregation of synucleins.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

CONVERSION OF TRYPSIN TO A COPPER ENZYME: TYROSINASE/CATECHOL OXIDASE BY CHEMICAL MODIFICATION

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu⁺²) introduced p-aminobenzamidine (pABA- Cu⁺²: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu⁺² preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K_cat, K_m, K_cat/K_m), optimum pH and temperature of the trypsin-pABA-Cu⁺² complex was also investigated.     

Stability and structure of binary and ternary complexes of alpha-lipoate and lipoate derivatives with Mn2+, Cu2+, and Zn2+ in solution.

The stabilities of the 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with lipoate and its chainshortened catabolites, viz., bisnorlipoate and tetranorlipoate, were studied by potentiometric titrations in water containing 50% dioxane (I = 0.1, NaClO₄; 25 °C). A comparison of the stabilities of these complexes with those of simple carboxylates reveals that the catabolite complexes formed with Cu²⁺ and Zn²⁺ are more stable than expected from only the basicity of the carboxylate groups. This is evidence that chelates involving the disulfide group are formed. The stability of all Mn²⁺ complexes is determined by the basicity of the carboxylate groups. The same pattern of stability holds for the mixed-ligand complexes formed by Cu²⁺ or Zn²⁺, 2,2′-bipyridyl, and lipoate or one of its derivatives. It is evident that the disulfide group of the 1,2-dithiolane moiety can participate in the formation of binary and ternary complexes. The somewhat less-pronounced coordinating properties of the 1,2-dithiolane moiety compared with the tetrahydrothiophene moiety are discussed. It is apparent that the electron density at S(1) and S(2) in the dithiolane moiety of lipoate is not equivalent: S(1) is favored over S(2) in electrophilic reactions; possible biological implications are indicated.     

Cu(II)–Protamine interaction. I. The formation and structure of Cu(II)–clupeine Z complexes

The formation and structure of four different Cu(II)–clupeine Z complexes have been studied using potentiometric and spectroscopic (ir and visible absorption, and CD) measurements. The results thus obtained indicate the presence of up to 8 binding sites in the pH range from 6.5 to 10.5. The spectroscopic evidence suggests that the strongest site available contains the α-amino terminal and the adjacent peptide nitrogen, which bind to copper from pH 5 to 6.5 to form the first complex. The stability constant of this first complex has a value of (9.5 ± 0.9) × 10³ mol^?1 1. From pH 6.5 to 8.5, two intervening guanidinium nitrogens of arginine residues occupy the two other corners of the coordination square, giving rise to the second complex. The other sites potentially available from pH 6.5 to 10.5 are formed by two amino nitrogens of arginine residues and two contiguous peptide nitrogens. The first intervene up to pH 8.5, forming the third complex, and the latter from this pH to 10.5, forming the fourth complex. Although the ligands intervening at sites 2–8 appear to be the same, the sites are by no means equivalent. The spectroscopic data enable one to distinguish three different types of binding sites.     

Metal ion interactions with mAbs: Part 1

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu²⁺, can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu²⁺-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu²⁺. More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu²⁺ mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu²⁺ induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.