A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Effect of dioxygen on copper(II) binding to α-synuclein

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (K_d(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M^∗DV(F/W), where M^∗ = methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a Cu^II-Trp/Phe (cation-π) interaction.     

Heterocyclic β-keto sulfide derivatives of carvacrol: Synthesis and copper (II) ion reducing capacity

Sixteen β-keto sulfide derivatives of carvacrol (4–19) incorporating phenyl or N, O and S heterocyclic moieties were synthesized in three steps. The relationships between heterocyclic structure and cupric, Cu(II), ion reducing antioxidant capacity (CUPRAC) were examined. Nine of the compounds (8–9 and 13–19) showed better CUPRAC activity than trolox at neutral pH, with trolox equivalent antioxidant capacity (TEAC) coefficients ranging between 1.20 and 1.75. Two derivatives (11–12) showed comparable reducing capacity to trolox, with TEAC values of 0.95 for 11 and 1.02 for 12. Compounds 8–9 and 11–19 were more effective at reducing the Cu(II) ion than ascorbic acid and the parent compound, carvacrol. The most effective antioxidants were those containing an oxadiazole, thiadiazole or triazole moiety. In particular, the methyl thiadiazole derivative (15) had the highest Cu(II) ion reducing capacity, with a TEAC coefficient of 1.73.     

Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal–Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal–protein interactions, including those in metal–Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu²⁺ binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10⁹ and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu²⁺-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu²⁺ binding to Aβ and other intrinsically disordered peptides.     

Computational study of binding of epothilone A to β-tubulin

Understanding the interactions of epothilones with β-tubulin is crucial for computer aided rational design of macrocyclic drugs based on epothilones and epothilone derivatives. Despite numerous structure-activity relationship investigations we still lack substantial knowledge about the binding mode of epothilones and their derivatives to β-tubulin. In this work, we reevaluated the electron crystallography structure of epothilone A/β-tubulin complex (PDB entry 1TVK) and proposed an alternative binding mode of epothilone A to β-tubulin that explains more experimental facts.     

Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.     

Copper(I) and copper(II) binding to β-amyloid 16 (Aβ16) studied by electrospray ionization mass spectrometry

Copper-β-amyloid 16 (Aβ16) complexes were investigated by electrospray ionization mass spectrometry (ESI-MS). Copper(i) and (ii) complexes were formed on-line in a microchip electrospray emitter by using a sacrificial copper electrode as the anode in positive ionization mode. In the presence of ascorbic acid in the peptide solution, the amount of Cu(i)-Aβ16 generated electrochemically was even higher. A kinetic model is proposed to account for the generation of copper complexes. The structure of Cu(i)-Aβ16 was investigated by tandem mass spectrometry (MS/MS), and the binding site of Cu(i) to Aβ16 was identified at the His13, His14 residues. Cu(ii)-Aβ16 was also investigated by MS/MS and, based on the unusual observations of a-ions, the two binding residues of His13 and His14 of Aβ16 to Cu(ii) were also confirmed. This approach provides direct information on Cu(i)-Aβ16 complexes generated in solution from metallic copper and gives evidence that both His13 and His14 are involved in the coordination of both Cu(i)- and Cu(ii)-Aβ16 complexes.     

The coordination of copper(II) with β-casomorphin and its fragments

The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H₂L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu₂L₂], bonded through the phenolic O^? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed.     

pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.     

Theoretical investigation of direct amination of β-ketoesters catalyzed by copper(II)-bisoxazoline(BOX)

The mechanism of the direct amination of β-keto esters catalyzed by copper(II)-bisoxazoline has been studied by means of density functional theory of B3LYP method. The computational results support the present mechanism, which involves (i) the generation of the enol from β-keto esters, which coordinates to copper(II)-bisoxazoline. The coordination step appears to be fast, exothermic, and irreversible. (ii) The formation of the σ(N-C) bond via a six-membered ring transition state after azo dicarboxylate coordination with the chiral catalyst. This step is chirality-control step. (iii) Intramolecular hydrogen migration generates a catalyst-product complex, which can finally yield product. The hydrogen shift is the rate-determining step, which affords the experimentally observed (R)-product. The stereochemical predictions have been rationalized in terms of steric repulsions, showing good agreement with experimental data. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Theoretical study of the hydroxylation of phenols mediated by an end-on bound superoxo–copper(II) complex

Peptidylglycine α-amidating monooxygenase and dopamine β-monooxygenase are copper-containing proteins which catalyze essential hydroxylation reactions in biological systems. There are several possible mechanisms for the reductive O₂-activation at their mononuclear copper active site. Recently, Karlin and coworkers reported on the reactivity of a copper(II)–superoxo complex which is capable of inducing the hydroxylation of phenols with incorporated oxygen atoms derived from the Cu(II)-O₂ ^·− moiety. In the present work the reaction mechanism for the abovementioned superoxo complex with phenols is studied. The pathways found are analyzed with the aim of providing some insight into the nature of the chemical and biological copper-promoted oxidative processes with 1:1 Cu(I)/O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of β-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of β-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.     

A tight coupling between β₂Y97 and β₂F200 of the GABA(A) receptor mediates GABA binding

The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α?R67, α?R120, α?R132, and β?R207) and two aromatic residues (β?Y97 and β?F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α?R67 nor α?R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α?R132 and both aromatic residues. Significant functional coupling between β?R207 and both β?Y97 and β?F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β?Y97 and β?F200, and for the first time provided direct evidence for the involvement of β?Y97 and β?F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.     

Structural and some medicinal characteristics of the copper(II)–hydroxychloroquine complex

In the first phase of this study, the binding of hydroxychloroquine to the copper(II) cation is examined using liquid chromatography–mass spectrometry (LC–MS), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), Fourier transform-ion cyclotron resonance spectrometry (FT-ICR) and nuclear magnetic resonance (¹H and ¹³C NMR) in one and two dimensions. The data suggest the metal–ligand complex is a polarity adaptive molecule. In the second phase of the study, the complexes activity is tested against the National Cancer Institute’s 60 cell line panel. Its anti-cancer activity is compared to quinine, Cu(II)–quinine and hydroxychloroquine. It serves as a base line for future anti-cancer complexes in which hydroxychloroquine is utilized for its ability to impact cell autophagy.     

Coordination chemistry of β-ketoamides: Synthesis of copper(II) complexes,x-ray structure of bis-(1-N-benzylamino-1,3-butanedionate)copper(II) and nucleophilic behavior of the metal-β-carbonylenolate ring toward cyanogen and benzoyl cyanide

N-monosubstituted β-ketoamides react almost quantitatively with copper(II) acetate in a 1/1 ethanol-water mixture at ca. 40 °C when acetic acid and solvent are removed at reduced pressure. The novel bis(β-ketoamidate)copper(II) complexes, which can be obtained with this synthetic procedure, are O,O′-bonded species, thermally stable both in the solid state and in solution of polar solvents. The crystal structure of bis(1-N-benzylamino-1,3-butanedionate)copper(II) has been determined. Crystals are orthorhombic, space group Pbca with Z = 4, in a unit cell of dimension a = 19.506(5),b = 11.901(4),c = 8.726(3)Å. The structure was solved by the heavy atom method and refined to R = 0.048 for 915 independent reflections. The complex is monomeric and no intermolecular Cu⋯O or Cu⋯N interactions are observed. The bis(benzoylacetanilidate)copper(II) complex reacts with cyanogen or benzoyl cyanide to give addition-insertion at the methino group of the metal-β-ketoamidate ring.     

Nickel(II)—bleomycin: spectral investigation of the metal binding site

Bleomycin (blm) solutions containing the nickel(II) ion have been investigated through ¹H nmr, ligand field and circular dichroism spectroscopies. It has been found the blm binds the metal ion in a pH dependent fashion. The spectral data are consistent with the presence of at least two species. It is suggested that in the low pH region blm binds to nickel(II) through the β-aminoalanino residue, whereas in the high pH region, the 4-amino-pyrimidine, imidazole, and amido group of β-hydroxy-histidine are also involved in coordination.     

Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase

Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.