Thermodynamic and structural characterization of the macrochelates formed in the reactions of copper(II) and zinc(II) ions with peptides of histidine

Copper(II) complexes of the peptides Ac-HisSarHis-NH₂, Ac-HisSarHisSarHis-NH₂ and Ac-HisSarHisSarHisSarHis-NH₂ have been studied by potentiometric, UV-Vis, CD and EPR spectroscopic methods. Stability constants for the corresponding zinc(II) complexes have also been reported. The formation of M(II)-2N_im, M(II)-3N_im and M(II)-4N_im bonded macrochelates was suggested in the pH range 5-7. The macrochelates were, however, not stable enough to prevent metal ion hydrolysis in slightly alkaline solutions. In the case of copper(II) complexes, the metal ion promoted deprotonation and coordination of the amide groups of histidyl residues were also suggested. The stability constants of macrochelate complexes were compared to the literature data reported for the macrochelates of the other peptides of histidine. It was found that the thermodynamic stability of macrochelate species is largely influenced by the number and location of histidyl residues in the peptide backbone. The highest stability was obtained for the HXHYH-type sequences, while the distant arrangement of histidyl residues resulted in a significant reduction of the stability constants.     

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH₂, L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH₂L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K_D,Cu = 7.4 × 10^− 13 M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH₂L is coordinated by four nitrogens {NH₂,3N_im} in the equatorial plane. In ZnH₂L the peptide binds to zinc(II) through a {NH₂,2N_im,COO⁻} donor set, and its zinc binding affinity is relatively modest (K_D,Zn = 4.8 × 10^− 7 M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH₂L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH₂L complexes has been verified by semiempirical quantum chemical calculations (PM6), too.     

N-terminal fragment of the anti-angiogenic human endostatin binds copper(II) with very high affinity

A histidine-rich peptide HSHRDFQPVLHL-NH₂ (L), identical with the N-terminal fragment of the anti-angiogenic human endostatin has been synthesized. Endostatin is a recently identified broad spectrum angiogenesis inhibitor, which inhibits 65 different tumor types. The N-terminal 25-mer peptide fragment of human endostatin has the same antitumor effect as the entire protein. The zinc(II) binding is crucial for the antitumor effect in both cases. Our peptide may provide all critical interactions for zinc(II) binding present in the N-terminal 25-mer peptide fragment. In addition, the N-terminus of human endostatin has a supposedly high affinity binding site for copper(II), similar to human serum albumin. Since copper(II) is intimately involved in angiogenesis, this may have biological relevance.In order to determine the metal binding properties of the N-terminal fragment of endostatin, we performed equilibrium, UV-visible (UV-vis), CD, EPR and NMR studies on the zinc(II) and copper(II) complexes of L. In the presence of zinc(II) the formation of a stable {NH₂, 3N_im, COO⁻} coordinated complex was detected in the neutral pH-range. This coordination mode is probably identical to that present in the zinc(II) complex of the above mentioned N-terminal 25-mer peptide fragment of human endostatin. Moreover, L has extremely high copper(II) binding affinity, close to those of copper-containing metalloenzymes, and forms albumin-like {NH₂, N⁻, N⁻, N_im} coordinated copper(II) complex in the neutral pH-range, which may suggest that copper(II) binding is involved in the biological activity of endostatin.     

Nickel(II) complexes of the multihistidine peptide fragments of human prion protein

Nickel(II) complexes of the peptide fragments of human prion protein containing histidyl residues both inside and outside the octarepeat domain have been studied by the combined application of potentiometric, UV-visible and circular dichroism spectroscopic methods. The imidazole-N donor atoms of histidyl residues are the exclusive metal binding sites below pH 7.5, but the formation of stable macrochelates was characteristic only for the peptide HuPrP(76-114) containing four histidyl residues. Yellow colored square planar complexes were obtained above pH 7.5-8 with the cooperative deprotonation of three amide nitrogens in the [N_im,N⁻,N⁻,N⁻] coordination mode. It was found that the peptides can bind as many nickel(II) ions as the number of independent histidyl residues. All data supported that the complex formation processes of nickel(II) are very similar to those of copper(II), but with a significantly reduced stability for nickel(II), which shifts the complex formation reactions into the slightly alkaline pH range. The formation of coordination isomers was characteristic of the mononuclear complexes with a significant preference for the nickel(II) binding at the histidyl sites outside the octarepeat domain. The results obtained for the two-histidine fragments of the protein, HuPrP(91-115), HuPrP(76-114)H85A and HuPrP(84-114)H96A, made it possible to compare the binding ability of the His96 and His111 sites. These data reveal a significant difference in the nickel(II) and copper(II) binding sites of the peptides: His96 was found to predominate almost completely for nickel(II) ions, while the opposite order, but with comparable concentrations, was reported for copper(II).     

The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center

In this paper are presented the features of copper (II) and zinc (II) heteronuclear complexes of the cyclic peptide—c(HKHGPG)₂. The coordination properties of ligand were studied by potentiometric, UV–Vis and CD spectroscopic methods. These experiments were carried out in aqueous solutions at 298 K depending on pH. It turned out that in a physiological pH dominates Cu(II)/Zn(II) complex ([CuZnL]⁴⁺) which could mimic the active center of superoxide dismutase (Cu,ZnSOD). In next step we performed in vitro research on Cu,ZnSOD activity for [CuZnL]⁴⁺ complex existing in 7.4 pH by the method of reduction of nitroblue tetrazolium (NBT). Also mono- and di-nuclear copper (II) complexes of this ligand were examined. The ability of inhibition free radical reaction were compared for all complexes. The results of these studies show that Cu(II) mono-, di-nuclear and Cu(II)/Zn(II) complexes becoming to new promising synthetic superoxide dismutase mimetics, and should be considered for further biological assays.     

Superoxide dismutase activity of macrocyclic polyamine complexes

Copper(II) and nickel(II) complexes of macrocyclic polyamine derivatives possessing partial oligopeptide-like structures are found to suppress the xanthine-xanthine oxidase-mediated reduction of nitroblue tetrazolium and also to suppress formazan formation by potassium superoxide. The activity in the superoxide dismutase assay is dependent on ring size, type and number of donor atoms, metal ion, and substituents on the macrocycles. Some of those are more active than the known O₂^? scavengers such as copper(II)-salicylate and copper(II)-amino acid (or peptide) complexes. Nickel (II)-naphthylmethyl-dioxo-[16]ane N₅, 13, 1 : 1 complex (NiH_?2L) is the most active among the 30 chelates examined.     

Superoxide dismutase–mimicking activities of dinuclear Cu(II) complexes with ligands containing a tetrathioether-tetraamino moiety

The superoxide scavenging activities of copper(II) complexes with the ligands, 6,6′-methylene-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L), and 6,6′-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L′), were investigated by xanthine–xanthine oxidase (X/XO) assays using nitroblue tetrazolium (NBT) as indicator molecule, and the results were compared with respect to the particular type of anion (ClO·4, Cl^·, NO·3) on the apical site of the copper(II) complexes. All of the complexes inhibited the reduction of NBT by superoxide radicals, with the [Cu₂(L′)](ClO₄)₂ complex exhibiting the highest scavenging activity against superoxide radicals among the complexes examined. The catalytic efficiency of the complexes for dismutation of superoxide radicals depends on the particular anion liganded to Cu(II) ion in the complexes, and the order of potency was observed to be ClO₄ > Cl > NO·3 in phosphate buffer at pH 7.40. The Cu(II)-H₄L′ complexes had the lowest IC₅₀ and catalytic rate constant values indicating that the distorted geometry of the Cu(II)-H₄L′ complexes influence their catalytic activities for dismutation of superoxide radicals more efficiently. The difference in the activities of the complexes toward superoxide radicals can also be attributed to the nature of the anions on the apical site of the copper(II) complexes and the superoxide dismutase-like activity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 53–59, 1998     

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

Theoretical analysis of intrinsic reaction kinetics and the behavior of immobilized enzymes system for steady-state conditions

Mathematical modeling of immobilized enzymes under different kinetics mechanism viz. simple Michaelis–Menten, uncompetitive substrate inhibition, total competitive product inhibition, total non-competitive product inhibition and reversible Michaelis–Menten reaction are discussed. These five kinetic models are based on reaction diffusion equations containing non-linear terms related to Michaelis–Menten kinetics of the enzymatic reaction. Modified Adomian decomposition method is employed to derive the general analytical expressions of substrate and product concentration for all these five mechanisms for all possible values of the parameters Φ_S (Thiele modulus for substrate), Φ_P (Thiele modulus for product) and α (dimensionless inhibition degree). Also we have presented the general analytical expressions for the mean integrated effectiveness factor for all values of parameters. Analytical results are compared with the numerical results and also with the limiting case results, which are found to be good in agreement.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

The structural and electrochemical consequences of hydrogenating copper N2S2 Schiff base macrocycles

A series of cis and trans tetradentate copper macrocyclic complexes, of ring size 14-16, that employ amine and thioether donor groups are reported. Apart from 5,6,15,16-bisbenzo-8,13-diaza-1,4-dithia-cyclohexadecane copper(I) (cis-[Cu(H₄N_buS_en)]⁺) all of the complexes are obtained in the copper(II) form. Crystallographic analysis shows that the copper(II) complexes all adopt a distorted planar geometry around the copper. In contrast, cis-[Cu(H₄N_buS_en)]⁺ is found to adopt a distorted tetrahedral geometry. The complexes were subjected to electrochemical analysis in water and acetonitrile. The effect of the solvent, positions of the donor atoms (cis/trans) on E^1/2 is discussed as is the comparison of the electrochemical behaviour of these complexes with their parent Schiff base macrocycles.     

Magnetic, spectroscopic, structural and biological properties of mixed-ligand complexes of copper(II) with N,N,N,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands

Two new mixed ligand complexes of copper(II) with N,N,N^′,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands have been prepared and characterized by means of spectroscopic, magnetic and single-crystal X-ray diffraction methods. These two complexes are isomorph and isostructure in which the coordination polyhedron about the copper(II) ion is distorted square pyramidal. [Cu(PMDT)(bipy)]²⁺ and [Cu(PMDT)(phen)]²⁺ show an absorption wavelength maximum at 625 and 678 nm, respectively, assigned to the d-d transition. Antibacterial, antifungal and superoxide dismutase activities of these complexes have also been measured. It was observed that [Cu(PMDT)(bipy)](ClO₄)₂ was more effective against P. Pyocyanea and Klebsiella sp. than S. aureus. Similarly, Fusarium sp. was highly susceptible against [Cu(PMDT)(bipy)](ClO₄)₂ but less susceptible against [Cu(PMDT)(phen)](ClO₄)₂.     

Copper(II) complexes of N2-alkyl-(S)-amino acid amides as chiral selectors for dynamically coated chiral stationary phases in RP-HPLC

Copper(II) complexes of N²-octyl-(S)-phenylalaninamide (Noc-Phe-NH₂), N²-dodecyl-(S)-phenylalaninamide (Ndo-Phe-NH₂), and N²-octyl-(S)-norleucinamide (Noc-NLeu-NH₂), dynamically adsorbed on a reversed-phase C₁₈ column, were able to perform the direct enantiomeric separation of unmodified amino acids, amino acid amides and esters, hydroxy acids, and dipeptides by elution with aqueous or mixed aqueous-organic solutions containing copper(II) sulphate or acetate. The role played by several parameters in the separation procedure was examined with the copper(II) complex of Noc-Phe-NH₂ [concentration of the copper(II) ion in the eluent, pH and eluent polarity, amount of adsorbed selector]. The separation was shown to occur entirely on the stationary phase. The mechanism of chiral discrimination is discussed in terms of the chromatographic parameters and of the structure of the copper(II) complexes in solution and in the solid state. The chiral stationary phase maintained its separation ability for about 3 months. However, the column could be easily restored by recovering the selector with methanol and repeating the loading procedure. © 1996 Wiley-Liss, Inc.     

Study of SOD Mimic and Nucleic Acid Interaction Activity Exerted by Enrofloxacin‐Based Copper(II) Complexes

Five new copper(II) complexes of type [Cu(erx)( L )Cl] (erx, enrofloxacin; thiophene‐2‐carbaldehyde ( L ¹ ); pyridine‐2‐carbaldehyde ( L ² ); 2,2′‐dipyridylamine ( L ³ ); 4,5‐diazafluoren‐9‐one ( L ⁴ ); bis(3,5‐dimethyl‐1‐pyrazolyl)methane ( L ⁵ )) have been synthesized and characterized by elemental analysis, reflectance, IR, and FAB‐MS. Complexes have been investigated for their interaction with calf thymus (CT) DNA utilizing the absorption‐titration method, viscometric and DNA thermal denaturation studies. The cleavage reaction on pUC19 DNA has been monitored by agarose gel electrophoresis. The results indicated that the Cu^II complexes can more effectively promote the cleavage of plasmid DNA at physiological pH and superoxide dismutase. The (SOD) activity of the complexes has been evaluated by the nitroblue tetrazolium assay, and the complexes catalyzed the dismutation of superoxide at pH 7.8 with IC₅₀ values of 0.35–1.25 μM . The complexes have also been screened for their antibacterial activity against five pathogenic bacteria.     

Copper(II) complexes of a new N-picolylated bis benzimidazolyl diamide ligand: Synthesis, crystal structure and catechol oxidase studies

Copper(II) complexes of a new bis benzimidazole diamide ligand N-picolyl-N,N′-bis(2-methylbenzimidazolyl)hexanediamide [Pic-GBHA = L₂] have been synthesized and characterized. One of the compound [Cu(L₂)(NO₃)₂] has been structurally characterized. The copper atom is bound to two benzimidazolyl nitrogen atoms, two amide carbonyl oxygen atoms and a bidentate nitrate ion, resulting in a distorted octahedral geometry. EPR spectra obtained at low temperature indicate a tetragonal geometry in the solution state. Complexes display a quasi-reversible redox wave due to the Cu(II)/Cu(I) reduction process having fairly cathodic E_1/2. These Cu(II) complexes were utilized to carry out oxidation of ditertbutylcatechol (DTBC) in methanol using molecular oxygen as the oxidant in. Low temperature EPR study of the oxidation reaction implicates the formation of an active copper species with fairly low A_‖ value. The presence of picolyl groups on the ligand also serve as a proton sponge giving 2-3 times higher rates of reaction in comparison to the non-picolylated ligand, implying a role of free basic groups in the pH control of enzymatic oxidation of catechols by catechol oxidase and tyrosinase.     

Coordination abilities of tetrapeptides containing proline and tyrosine—A spectrophotometric and potentiometric study

The synthesis of three tetrapeptides, Gly-Pro-Gly-Tyr. Gly-Pro-Tyr-Gly. and Tyr-Pro-Gly-Gly, are described. All contain proline as the second amino acid subunit to act as a break point in metal complex formation.The proton and copper(II) complex formation constants have been measured at 22°C and l = 0.10 mol dm^?3 (KNO₃). The copper(II) complexes have also been studied spectrophotometrically over the pH range of 6–11 by absorption spectroscopy (800–200 nm), circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy. The experimental data have been combined to determine the complex species present as a function of pH and the coordination centers used.     

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N⁵-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coli N⁵-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis–Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis–Menten kinetics. Instead, these compounds inhibit N⁵-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N⁵-CAIR synthetase.     

The galactose biosensor based on microporous polyacrylonitrile

A galactose biosensor is obtained by immobilizing galactose oxidase (GAO) in a microporous polyacrylonitrile (PAN) thin film. The effects of pH, potential and temperature on response current are studied. The optimum pH and apparent activation energy of enzyme-catalyzed reaction are 7.1 and 31.1?kJ?mol^?1, respectively. The response current of the biosensor increases linearly with the increasing galactose concentration from 0.02 to 1.60?mmol?dm^?3. The Michaelis–Menten constant value (K_m^app) is 12.15?mmol?dm^?3. The biosensor shows good operational stability and reproducibility. The galactose biosensor is characterized with cyclic voltammogram, FTIR and UV-Vis.     

Decomposition of reactive oxygen species by copper(II) bis(1-pyrazolyl)methane complexes

Two bis(1-pyrazolyl)alkane ligands, bis(3,5-dimethyl-1-pyrazolyl)methane and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methane, and their copper(II) complexes, bis(3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₁(NO₃)₂] and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₂(NO₃)₂]·2H₂O, were prepared. Physiochemical properties of the copper(II) complexes were studied by spectroscopic (UV–vis, IR, EPR) techniques and cyclic voltammetry. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:copper(II) ion and a bindentate coordination mode for the nitrate ions in both of the complexes. According to experimental and theoretical ab initio data, the copper(II) ion is located in an octahedral hexacoordinated environment. Both complexes were able to catalyze the dismutation of superoxide anion () (pH 7.5) and decomposition of H₂O₂ (pH 7.5) and peroxynitrite (pH 10.9). In addition, both complexes exhibited superoxide dismutase (SOD) like activity toward extracellular and intracellular reactive oxygen species produced by activated human neutrophils in whole blood. Thus, these complexes represent useful SOD mimetics with a broad range of antioxidant activity toward a variety of reactive oxidants.     

Study of copper(II) and nickel(II) ternary complexes involving tertiary amines and phenyl or hydroxylphenyl substituted amino acids

Formation constants of ternary complexes MAL, where M = Cu(II) or Ni(II). A = 2.2′bipyridyl. 1, 10-phenanthroline, and L = 3.4-dihydroxyphenylalanine (dopa), tyrosine, or phenylalanine have been determined by using the computer program SCOGS. It is observed that dopa coordinates with Cu(II)-A and Ni(II)-A through the aminocarboxylate and only over the pH range 3–8, though the ligand coordinates with free Cu(II) ion from the amino carboxylate end in the lower pH range (pH 2–4) and from the catechol end at the higher pH range (pH > 5). The visible spectrum of Cu-A-dopa is similar to that of Cu-A-phenylalanine or Cu-A-tyrosine over the entire pH range, confirming amino carboxylate coordination. Δ log K (K_MAL - log_KML) is found to be positive in all the six Cu(II) complexes. whereas it is negative in Ni(II) complexes. Release in the ternary complexes of the repulsion between the Cu(II) dπ electron and electrons delocalized over the phenyl ring has been proposed as a probable reason for the positive Δ log K.