Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations

Abstract Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo' -deficient mutants of Escherichia coli K-12 with the following order of potency: Zn ( II ) > Cd ( II ) > > Co ( II ). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.     

Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1

In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-β-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a k_cat of 63 s⁻¹ and K_m of 20 μM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III).     

A ratiometric fluorescent detection of Zn(II) in aqueous solutions using pyrene-appended histidine

A fluorescent chemosensor, Py-His, based on histidine was easily synthesized in solid phase synthesis. Py-His displayed a highly sensitive ratiometric response to Zn(II) with potent binding affinity (K_a = 1.17 × 10¹³ M^?2) in aqueous solutions. The detection limit of Py-His for Zn(II) was calculated as 80.8 nM. Moreover, Py-His distinguished Zn(II) and Hg(II) by different ratiometric response type; the chemosensor showed a more enhanced increase of excimer emission intensity to Zn(II) than Hg(II). Upon addition of Ag(I) and Cu(II), Py-His showed a turn-off response mainly due to the quenching effect of these metal ions. The binding stoichiometry (2:1 or 1:1) of Py-His to target metal ions played a critical role in the fluorescent response type (ratiometric and turn off response) to target metal ions. The role of imidazole group of Py-His for ratiometric detection of Zn(II) was proposed by pH titration experiments.     

Zinc(II) complexation by some biologically relevant pH buffers

The isothermal titration calorimetry (ITC) technique supported by potentiometric titration data was used to study the interaction of zinc ions with pH buffer substances, namely 2‐(N‐morpholino)ethanesulfonic acid (Mes), piperazine‐N,N′‐bis(2‐ethanesulfonic acid) (Pipes), and dimethylarsenic acid (Caco). The displacement ITC titration method with nitrilotriacetic acid as a strong, competitive ligand was applied to determine conditional–independent thermodynamic parameters for the binding of Zn(II) to Mes, Pipes, and Caco. Furthermore, the relationship between the proposed coordination mode of the buffers and the binding enthalpy has been discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Metal complexes of naphthoquinone based ligand: synthesis,characterization, protein binding,DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (10⁵ M^?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (K_app 10⁵ M^?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.     

A switch‐off fluorescence probe towards Pb(II) and cu(II) ions based on a calix[4]pyrrole bearing amino‐quinoline group

A new fluorescence receptor calix[4]pyrrole‐N‐(quinoline‐8‐yl) acetamide (CAMQ) containing a pyrrolic ring connected via the meso‐position was synthesized, purified and characterized by elemental analysis, NMR and mass spectroscopy. This compound was examined for its fluorescence properties towards different metal ions e.g. Ag(I), Hg(II), Co(II), Ca(II), Ni(II), Zn(II), Cr(II), Ba(II), Fe(II), Cu(II), Pb(II)and Mg(II) ions by spectrophotometry and spectrofluorometry. It was concluded that the compound (CAMQ) possessed significantly enhanced selectivity for Pb(II) and Cu(II) ions in dimethyl sulfoxide (DMSO) even at very low concentrations (1 μM). It exhibit ‘turn‐on’ fluorescence when exposed to Pb(II) and Cu(II) and did so in preference to other metal ions. The binding constants, stoichiometry and quantum yields have been determined. The quenching mechanism was assessed using the Stern–Volmer equation and was also discussed.     

Synthesis and multi‐spectroscopic study on DNA‐binding,cleavage and biological properties of M(II) complexes based on N2O2 donor Schiff base ligand

A novel Schiff base, (S,Z)‐4‐(methylthio)‐2‐((3‐oxo‐2,3‐dihydro‐1H‐inden‐1‐ylidene)amino)butanoic acid (L) and four M(II) complexes (where M = Co, Cu, Ni and Zn) were synthesized and characterized. The DNA‐binding characteristics of the complexes were investigated using various spectroscopic methods and viscosity measurements. Analysis of the results suggests that all the complexes bind to calf thymus DNA via intercalation. Among the four, Cu(II) complex was found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365 nm. These complexes also exhibit good antioxidant activities against 2,2‐diphenyl‐1‐picrylhydrazyl radical. In vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents.     

Synthesis and characterisation of anthracene-based fluorophore and its interactions with selected metal ions

An anthracene-based novel ligand (L), 9,10-bis((4,6-dimethylpyrimidin-2-ylthio)methyl)anthracene, was synthesised and fully characterised. Interactions of the ligand with selected metal ions, Hg(II), Cu(II), Ag(I), Pb(II), Zn(II), Ni(II), Co(II), and Cr(III), were spectroscopically investigated. Of the examined metal ions, both Hg(II) and Cu(II) showed responses in both UV-Vis and fluorescent spectroscopy towards the ligand in acetonitrile solution. Spectroscopic titration indicated that the ligand forms complexes with the two metal ions in 1:1 and 1:2 ratios, respectively. DFT calculations revealed that Hg(II) binds possibly with two pairs of donor-set {SN} of the ligand to form a mononuclear complex in a distorted planar geometry whereas Cu(II) forms likely a binuclear complex in a tetrahedral geometry in which each Cu(II) is further coordinated with possibly two acetonitrile molecules.     

Variations in the coordination environment of Co, Cu and Zn complexes prepared from a tridentate (imino)pyridine ligand and their structural comparisons

The variations in the coordination environment of Co(II), Cu(II) and Zn(II) complexes with the neutral, tridentate ligand bis[1-(cyclohexylimino)ethyl]pyridine (BCIP) are reported. Analogous syntheses were carried out utilizing either the M(BF₄)₂ · xH₂O or MCl₂ · xH₂O metal salts (where M = Co(II), Cu(II) or Zn(II)) with one equivalent of BCIP. When the hydrated metal starting material was used, cationic, octahedral complexes of the type [M(BCIP)₂]²⁺ were isolated as the tetrafluoroborate salt (4, 5). Conversely, when the hydrated chloride metal salt was used as the starting material, only neutral, pentacoordinate [M(BCIP)Cl₂] complexes (1-3) formed. All complexes were characterized by X-ray diffraction studies. The three complexes that are five coordinate have distortions due mainly to the pyridine di-imine bite angle. The [Cu(BCIP)Cl₂] (2) also exhibits deviations in the Cu(II)-Cl bond distances with values of 2.4242(9) and 2.2505(9) Å, which are not seen in the analogous Zn(II) and Co(II) structures. Similarly, the two six coordinate complexes (5, 6) are also altered by the ligand frame bite angle giving rise to distorted octahedral geometries in each complex. The [Cu(BCIP)₂](BF₄)₂ (6) also exhibits Cu(II)-N_imine bond lengths that are on average 0.14 Å longer than those found in the analogous 5 coordinate complex, [Cu(BCIP)Cl₂]. In addition to X-ray analysis, all complexes were also characterized by UV/Vis and IR spectroscopy with ¹H NMR spectroscopy being used for the analysis of the Zn(II) analogue (3).     

Targeting topoisomerase II with the chiral DNA-intercalating ruthenium(II) polypyridyl complexes

Many antitumor drugs act as topoisomerase inhibitors, and the inhibitions are usually related to DNA binding. Here we designed and synthesized DNA-intercalating Ru(II) polypyridyl complexes Δ--[Ru(bpy)₂(uip)]²⁺ and Λ-[Ru(bpy)₂(uip)]²⁺ (bpy is 2,2′-bipyridyl, uip is 2-(5-uracil)-1H-imidazo[4,5-f][1,10]phenanthroline). The DNA binding, photocleavage, topoisomerase inhibition, and cytotoxicity of the complexes were studied. As we expected, the synthesized Ru(II) complexes can intercalate into DNA base pairs and cleave the pBR322 DNA with high activity upon irradiation. The mechanism studies reveal that singlet oxygen (¹O₂) and superoxide anion radical (O₂^•−) may play an important role in the photocleavage. The inhibition of topoisomerases I and II by the Ru(II) complexes has been studied. The results suggest that both complexes are efficient inhibitors towards topoisomerase II by interference with the DNA religation and direct topoisomerase II binding. Both complexes show antitumor activity towards HELA, hepG2, BEL-7402, and CNE-1 tumor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Factors affecting nucleolytic efficiency of some ternary metal complexes with DNA binding and recognition domains. Crystal and molecular structure of Zn(phen)(edda)

The binding selectivity of the M(phen)(edda) (M = Cu, Co, Ni, Zn; phen = 1,10-phenanthroline, edda = ethylenediaminediacetic acid) complexes towards ds(CG)₆, ds(AT)₆ and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(II) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N₄O₂ octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that inter-digitate with adjacent double layers via π…π interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling.     

Biological evaluations of newly-designed Pt(II) and Pd(II) complexes using spectroscopic and molecular docking approaches

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7?×?10³ M^?1, 2.65?×?10³ M^?1, 0.3?×?10³ M^?1, and 4.4?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25?°C, respectively. Also, binding constants of 1.9?×?10³ M^?1, 15.17?×?10³ M^?1, 1.9?×?10³ M^?1, and 13.1?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37?°C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.

Communicated by Ramaswamy H. Sarma     

Physiological levels of glutathione enhance Zn(II) binding by a Cys4 zinc finger

Using fluorescence and UV-vis spectroscopies and mass spectrometry, we demonstrated that the presence of physiological levels of reduced glutathione enhances the binding of Zn(II) to XPAzf, a Cys4 zinc finger peptide derived from the XPA protein, by means of formation of a ternary complex of a general formula ZnXPAzf[GSH]. Similar complexes were also indicated by ESI-MS for isostructural Co(II)- and Cd(II)-substituted XPAzf. The observed enhancement of the Zn(II) binding to XPAzf by a factor of 50 over the physiological range of GSH concentrations of 1-20 mM corresponds to a dissociation constant of GSH from the ZnXPAzf[GSH] complex of 0.05 μM. This effect may account for an apparent discrepancy between relatively low Zn(II) binding constants measured in vitro for many zinc fingers, and the requirement of tight Zn(II) binding enforced by intracellular zinc buffering by the thionein/metallothionein couple.     

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO₃ (I) and [Zn(phen)(Gly)]NO₃ (II) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV–vis, FT-IR, and ¹H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV–vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV–vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow’s site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.     

Synthesis,cytotoxicity and DNA-binding properties of Pd(II), Cu(II) and Zn(II) complexes with 4′-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-2,2′:6′,2″-terpyridine

Pd(II), Cu(II) and Zn(II) complexes (1–3) based on 4′-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-2,2′:6′,2″-terpyridine were synthesized and characterized by UV, IR, NMR, EPR, HRMS, elemental analyses, and molar conductivity measurements. The cytotoxicity of these complexes against HL-60, BGC-823, KB, Bel-7402, A549, Hela, K562 and MCF-7 cell lines in vitro was measured by MTT method. The DNA binding property of the complexes was evaluated by UV, fluorescence, CD spectroscopies and thermal denaturation. The cytotoxicity of complexes 1 and 3 against all the tested cell lines is better than that of cisplatin. Complexes 1 and 2 exhibit 7- and 4-folds higher cytotoxicity than cisplatin against Bel-7402 cell line. Complex 3 displays the highest cytotoxicity against all the cell lines tested, and shows 7-, 14-, 8-, 11- and 8-folds higher cytotoxicity than cisplatin against Bel-7402, A549, Hela, K562 and MCF-7 cell lines. The complexes bind to DNA via intercalation mode and complex 3 stabilizes the G-quadruplex. The results reveal that all the complexes display high cytotoxicity against all the tested cancer cell lines, and complex 3 is selective for G-quadruplex over duplex DNA.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV–Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K_b = 1.4 × 10⁴ M^?1) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 ?4.8 × 10⁴ M^?1. CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and Van der Waals play main roles in this binding prose. Competitive fluorimetric studies with methylene blue (MB) dye have shown that Zn(II) complex exhibits the ability of this complex to displace with DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.     

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 10⁴ M⁻¹, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.