The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.     

Cobalt(II) and zinc(II) binding to a ferredoxin maquette

We have examined the Co(II) and Zn(II) affinity of the prototype ferredoxin maquette ligand, NH(2)-KLCEGG.CIACGAC.GGW-CONH2 (IAA), which was originally designed to bind a [4Fe-4S] cluster. UV-Vis spectroscopy demonstrates tight 1:1 complex formation between Co(II) and IAA. The intensity of the S-->Co(II) charge transfer bands at 304 and 340 nm and the ligand field bands between 630 and 728 nm indicate Co(II) coordination by the four cysteine thiolates of IAA in a pseudo-tetrahedral geometry. A dissociation constant value of 5.3 microM was determined for the Co(II)-IAA complex at pH 6.5. Zn(II) readily displaces Co(II) from IAA as evinced by loss of the Co(II) spectral features. The dissociation constant for Zn(II), 20 pM at pH 6.5, was determined be competition experiments with Co(II)-IAA. These results demonstrate that the ferredoxin maquette ligand is an excellent ligand for Zn(II).     

The binding of zinc (II) to a double-stranded oligodeoxyribonucleotide. A voltammetric study

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62 x 10(-9) to 3.62 x 10(-8) M and 4.06 x 10(-10) to 5.25 x 10(-9) M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation-reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v(1/2) (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide.     

The binding of zinc(II) to Mung Bean Nuclease. A voltammetric study

Binding of zinc to Mung Bean Nuclease was investigated by anodic stripping voltammetry and cyclic voltammetry. These methods rely on the direct monitoring of the oxidation current of zinc in the absence and presence of Mung Bean Nuclease. Titration curves of Zn(2+) with the enzyme were obtained in concentrations ranging from 1.08x10(-9) to 1.07x10(-8) M and 1.16x10(-8) to 1.04x10(-7) M. The acquired data were used to calculate the dissociation constant and the stoichiometry of the complex. The binding sites of zinc in the Mung Bean Nuclease molecule were investigated using cyclic voltammetry. Two types of binding sites for zinc were identified and were attributed to a mononuclear exposed zinc-binding site with catalytic function and to an inaccessible binuclear zinc-binding site with structural functions.     

The crystallochemistry of tetracyanocomplexes. The crystal and molecular structures of tris(1,2-diaminoethane)zinc(II) tetracyanoniccolate(II) monohydrate and tris(1,2-diaminoethane)zinc(II) tetracyanoniccolate(II)

C₁₀H₂₆N₁₀ONiZn, tris(1,2-diaminoethane) zinc(II) tetrakis(cyano)niccolate(II) monohydrate (I), orthorhombic, Pbca, a = 1.1680(4), b = 1.5844(3), c = 1.9981(6) nm, Z = 8 d(meas) = 1.54, d(calc) = 1.53 g cm^?3. C₁₀H₂₄N₁₀NiZn, tris(1,2-diaminoethane) zinc(II) terakis(cyano)niccolate(II), (II), monoclinic, P2₁/n, a = 0.7957(2), b = 1.5170(5), c = 1.4932(4) nm, β = 96.41(2)°, Z = 4, d(meas) = 1.49, d(calc) = 1.51 g cm^?3. Both the structures (I) and (II) have been solved by the heavy atom method and refined by full-matrix least-squares to R(I) = 0.086 for 1890 independent reflections and R(II) = 0.058 for 1689 independent reflections, respectively. In the case of (II) the superlattice structure problem was solved. The crystal structure of (I) consists of [Zn(en)₃]²⁺ cations, [Ni(CN)₄]^2? anions and water molecules. Two of the cyano groups in trans positions are bonded to water molecules by hydrogen bonds, the distances CN?O being 0.289 and 0.291 nm, respectively. The crystal structure of (II) is constituted by [Zn(en)₃]²⁺ cations and [Ni(CN)₄]^2? anions.     

An atomistic view of the YiiP structural changes upon zinc(II) binding

BackgroundYiiP is a bacterial zinc-for-proton antiporter belonging to the cation diffusion facilitator family. The zinc(II) ions are transported across the cell membrane, from the cytosol to the extracellular space.MethodsWe performed atomistic molecular dynamics simulations of the YiiP dimer with zinc(II) ions in solution to elucidate how the metal ions interact with the protein while moving from the cytosol to the transport site.ResultsWe observed that of the two cavities of the dimer, only one was accessible from the cytosol during transport. Zinc(II) binding to D49 of the transport site triggered a rearrangement of the transmembrane domain that closed the accessible cavity. Finally, we analyzed the free-energy profiles of metal transit in the channel and observed the existence of a high barrier preventing release from the transport site.ConclusionsThe observed dynamics is consistent with the dimer-dimer interface forming a stable scaffold against which the rest of the trans-membrane rearranges.General significanceZinc(II) transporters are present in all kingdoms of life. The present study highlights structural features that might be of general relevance.     

Selective binding behavior of zinc(II) and copper(II) ions to their native sites of apo-bovine superoxide dismutase

The four binding constants of zinc(II) ions to apo-bovine superoxide dismutase were measured by the method of equilibrium dialysis. The binding constants (10(11.1)-10(10.9) M-1) of zinc ions to the native zinc sites were much larger than those to the native copper sites (10(7.8)-10(6.5) M-1) at pH 6.25. The competitive reaction between copper(II) and zinc(II) ions for the native copper sites of copper free bovine superoxide dismutase was also investigated. The native copper sites of bovine superoxide dismutase selectively react with copper ions, because the binding constants of copper ions for the native copper sites were much larger (10(6) times) than those of zinc ions.     

The use of zinc(II) to probe iron binding and oxidation by the ferritin (EcFtnA) of Escherichia coli

 The ferritin of Escherichia coli (EcFtnA) is similar to human H-chain ferritin (HuHF) in having 24 subunits, each containing a dinuclear site at which two iron atoms can be oxidised (the diiron centre). In EcFtnA, unlike HuHF, fluorescence quenching of Trp122, located near site A of the dinuclear centre, can be used to monitor metal binding (this tryptophan is absent from HuHF). Metal binding also perturbs the UV absorbance spectrum of Trp122 and that of Tyr24 (a conserved residue near site B of the dinuclear centre). Using UV-difference spectroscopy and fluorescence quenching it is shown that Fe(II) and Zn(II) bind at the same sites, A and B. Sequential stopped-flow studies of Fe(II) binding and oxidation also show that Zn(II) is an effective competitor of Fe(II) binding and an inhibitor of its oxidation. Received: 10 June 1998 / Accepted: 18 September 1998     

The binding of Mg(II) and Ni(II) to synthetic polynucleotides

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.     

Equilibrium studies of zinc(II) and cobalt(II) binding to tripeptide analogues of the amino terminus of human serum albumin

Serum albumin is known to bind several divalent metal ions at the amino terminus of the protein. Two peptide analogues for the amino terminus of human albumin, L-aspartyl-L-alanyl-L-histidine-N-Methyl amide (AAHNMA) and glycylglycyl-L-histidine-N-methyl amide (GGHNMA) have been synthesized, and their interactions with Zn(II) and Co(II) ions have been studied using analytical potentiometry. The stability constants of the species and their distribution as a function of pH were determined in 0.16-M KNO₃ at 25°. Comparison of the modes of interaction of the Zn(II) and Co(II) with each of the above peptides indicate that, although Co(II) is a valuable tool for the study of Zn(II) interaction with metalloenzymes, it is considerably less useful as a Zn(II) model with small peptide molecules. The potentiometric properties of the two peptide-Zn(II) systems have been compared to the potentiostatic properties of the albumin-Zn(II) system. The results indicate that AAHNMA is a better analogue for the Zn(II)-HSA interaction than is GGHNMA. The findings suggest that the Zn(II)-HSA binding site is best described as a compound site containing both a histidyl and a neighboring carboxyl group.     

X-ray diffraction study of the zinc(II) binding sites in yeast phenylalanine transfer RNA preferential binding of zinc to guanines in purine-purine sequences

An X-ray diffraction study of a zinc(II) complex of tRNA^Phe from yeast reveals the presence of five zin-binding sites on the tRNA molecule. Two of the cooperatively bound Mg²⁺ in the native tRNA structure are replaced by Zn²⁺. The remaining sites involve direct coordination of zinc to the N7 position of tRNA guanine bases, G15, G43 and G45. Thus, zinc displays a high specificity for binding to guanine bases in purine-purine sequences.     

3,5-Lutidine coordinated zinc(II) aryl carboxylate complexes: Precursors for zinc(II) oxide

The reaction of Zn(κ²O,O′-OAc)₂·2H₂O with two equiv of 3,5-lutidine in methanol at room temperature for 12 h afforded [Zn(OAc)₂(3,5-lutidine)₂]· H₂O (1) in 91% yield. The acetate exchange reaction of 1 with two equiv of aryl carboxylic acids in methanol at room temperature for 12 h afforded [Zn(μ₂-κ¹O:κ¹O′-O₂CAr)₂(3,5-lutidine)]₂ [Ar = C₆H₅ (2) and C₆H₄Me-3 (3)], [Zn(OC(O)C₆H₄Me-2)₂(3,5-lutidine)₂] (4) and [Zn(κ²O,O′−O₂CC₆H₄Me-4)₂(3,5-lutidine)₂] (5) in ?90% yield. Complexes 1-5 were characterized by microanalytical, IR, solution (¹H and ¹³C) and solid-state cross-polarization magic angle spinning ¹³C NMR and X-ray diffraction data. The zinc atom in 1 is surrounded by nitrogen atom of two 3,5-lutidine and oxygen atom of two monodentate acetate moiety and thus attains a tetrahedral geometry. One of the acetate moieties is hydrogen bonded with a water molecule in the crystal lattice. Complexes 2 and 3 possess a dinuclear paddlewheel framework with a square pyramidal geometry around the zinc atom whereas 4 and 5 are mononuclear species with the zinc atom in tetrahedral and an octahedral geometry, respectively. Thermogravimetric analyses of 2-5 suggested ZnO as the decomposed product followed by the confirmation from the powder X-ray diffraction patterns. Enormous gas evolution resulting in porous ZnO during thermal decomposition was evidenced from scanning electron microscopic images.     

Modulation of the zinc(II) center in protein farnesyltransferase by mutagenesis of the zinc(II) ligands

Protein farnesyltransferase (PFTase) is a zinc-containing metalloenzyme that catalyzes the alkylation of cysteine (C) in protein substrates containing a C-terminal "CaaX" motif by farnesyl diphosphate (FPP). In yeast PFTase Zn(II) is coordinated to D307, C309, and H363 in the beta-subunit. The inner coordination sphere of the metal also contains a water molecule to give a net charge of 0 for the tetracoordinate Zn(II) center. When the protein substrate binds, the water molecule is replaced by the thiol of the cysteine residue, and the thiol is deprotonated to generate a Zn(II)-stabilized thiolate in the PFTase.FPP.protein ternary complex for the ensuing prenyl transfer reaction. An expression system was constructed for yeast PFTase containing a His(6) tag at the C-terminus of the beta-subunit to facilitate purification of the wild-type enzyme and site-directed mutants. The amino acids that coordinate Zn(II) were substituted to give a series of mutant PFTases with net charges of +1, 0, -1, and -2 at the Zn(II) center of the ternary enzyme.substrate complexes. Wild-type PFTase and the site-directed mutants were purified as alpha,beta-heterodimers, and each was found to contain an equivalent of Zn(II). All of the mutants were less reactive than wt PFTase (net charge of -1), with the greatest losses of activity seen for the mutants with net charges of 0 and +1. Equilibrium binding experiments with dGCVIA peptide and an unreactive analogue of FPP, (E,E)-2-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl]phosphonate (FNP), established that all of the mutants bound an equivalent of the peptide substrate. Like wt PFTase, the pH dependence of K(D) for the mutants did not change significantly between pH 5 and pH 9, indicating that pK(A)s for the thiol moiety in the (mutant PFTase).FNP.peptide complexes were <5. dGSVIA and dG(beta-NH2-Ala)VIA, where the sulfhydryl moiety was replaced by hydroxyl and amino groups, respectively, were not substrates. These experiments suggest a direct relationship between the net charge of the Zn(II) center in PFTase and the reactivity of the peptide thiolate that is alkylated by FPP.     

Chemistry of zinc(II) fluorophore sensors

The biological role of the zinc(II) ion has been recognized in DNA and RNA synthesis, apoptosis, gene expression, or protein structure and function. Therefore, development of useful zinc(II) sensors has recently been attracting much interest. Chemistry for selective and efficient detection of trace Zn²⁺ is a central issue. Recently, various types of zinc-fluorophores are emerging, comprising bio-inspired aromatic sulfonamide derivatives, zinc-finger peptides attached to fluorescent dyes, or fluorophore-pendant macrocyclic polyamines. The chemical principles, properties and limitations of these Zn²⁺-fluorophores are discussed.     

DNA binding affinity and antioxidative activity of copper(II) and zinc(II) complexes with a novel hesperetin Schiff base ligand

A hesperetin Schiff base ligand (H₄L) and its complexes, [H₃CuL·OAc]·H₂O and [H₃ZnL·OAc]·2H₂O, have been synthesized and characterized on the basis of elemental analysis, molar conductivity, ¹H NMR, mass spectra, UV-Vis spectra and IR spectra. The binding of these two complexes and the ligand to DNA has been investigated by ultraviolet absorption spectroscopy, fluorescence spectroscopy and viscosity measurements. The experiments indicate that all the compounds can bind to DNA through an intercalative mode and the complexes intercalate into DNA more deeply than that of the ligand. In addition, the antioxidative activity was also determined. The 50% inhibition obtained for the ligand and its complexes demonstrates that, compared to the ligand, the complexes exhibit higher antioxidative activity in the suppression of and HO.     

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.