Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin

The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (approximately 9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.     

Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions

Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole/quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK(a) between 9 and 13, and quinone imines (QI), with pK(a) of 6.3. The presence of the quinone-imine tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK(a) 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV-Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat)(-) and Cu(QI)(Cat)(-) are shown to dominate at physiological pH.     

Complexes of Ni(II) and Cu(II) with ofloxacin. Crystal structure of a new Cu(II) ofloxacin complex

Several coordination compounds formed between Ni(II) or Cu(II) with ofloxacin have been synthesised and characterised. According to elemental chemical analysis and FT-IR spectroscopy data, direct reaction of Ni(II) and Cu(II) salts with ofloxacin leads to formation of precipitates for which mass spectrometry demonstrates their polymeric nature. However, crystalline [Cu(oflo)2(H2O)].2H2O is formed if the reaction is carried out in the presence of ammonia. This complex crystallises in the triclinic system, space group P-1 with a=9.2887(12), b=11.2376(14), c=17.874(2) A, alpha=92.12(3), beta=95.39(3), gamma=91.71(3) degrees and Z=2. The local geometry around the Cu(II) ion is a slightly distorted square base pyramid. Electronic spectra, magnetic susceptibility measurements and EPR spectra of the synthesised complexes indicate a tetragonal environment.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath)

We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.     

Cu(II) binding to monomeric, oligomeric, and fibrillar forms of the Alzheimer's disease amyloid-beta peptide

Copper has been proposed to play a role in Alzheimer's disease through interactions with the amyoid-beta (Abeta) peptide. The coordination environment of bound copper as a function of Cu:Abeta stoichiometry and Abeta oligomerization state are particularly contentious. Using low-temperature electron paramagnetic resonance (EPR) spectroscopy, we spectroscopically distinguish two Cu(II) binding sites on both soluble and fibrillar Abeta (for site 1, A parallel = 168 +/- 1 G and g parallel = 2.268; for site 2, A parallel = 157 +/- 2 G and g parallel = 2.303). When fibrils that have been incubated with more than 1 equiv of Cu(II) are washed, the second Cu(II) ion is removed, indicating that it is only weakly bound to the fibrils. No change in the Cu(II) coordination environment is detected by EPR spectroscopy of Cu(II) with Abeta (1:1 ratio) collected as a function of Abeta fibrillization time, which indicates that the Cu(II) environment is independent of Abeta oligomeric state. The initial Cu(II)-Abeta complexes go on to form Cu(II)-containing Abeta fibrils. Transmission electron microscopy images of Abeta fibrils before and after Cu(II) addition are the same, showing that once incorporated, Cu(II) does not affect fibrillar structure; however, the presence of Cu(II) appears to induce fibril-fibril association. On the basis of our results, we propose a model for Cu(II) binding to Abeta during fibrillization that is independent of peptide oligomeric state.     

EPR studies of copper(II) and cobalt(II) complexes of adriamycin

Cu2+ and Co2+ complexes of adriamycin (ADM) in aqueous solutions have been examined using EPR spectroscopy. An appreciable amount of Cu2+ and Co2+ complexes formed in the solutions were found to be in the EPR silent associated form, where the metal ions are antiferromagnetically coupled. The associated form of the Cu2+ complex may be neither a simple dimer nor coordination polymer but aggregates of a stacked type. Formation of a complex having Cu2+-ADM stoichiometry of 1:2 was observed for the solutions containing excess of ADM as an EPR observable species. The complex having Cu2+-ADM stoichiometry of 1:1 was not observed directly by EPR, but the presence of the complex is undeniable, especially at low pH range so far as large excessive ADM is not present. The Co2+ complex of ADM observed by EPR is in the high-spin (S = 3/2) state and may have a coordination structure of tetragonal symmetry. The EPR spectra of these complexes apparently show that the Cu2+ and Co2+ ions are bound at the carbonyl and phenolate oxygen in the 1,4-dihydroxyanthraquinone moiety and the amino nitrogen in the sugar part does not seem to participate in the coordination to the metal ions.     

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.     

Empirical rules for rationalising visible circular dichroism of Cu2+ and Ni2+ histidine complexes: applications to the prion protein

A natively unfolded region of the prion protein, PrP(90-126) binds Cu(2+) ions and is vital for prion propagation. Pentapeptides, acyl-GGGTH(92-96) and acyl-TNMKH(107-111), represent the minimum motif for this Cu(2+) binding region. EPR and (1)H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His(96) and His(111) to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu(2+) visible CD spectra involving histidine and amide main-chain coordination.     

A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH: relevance to the fifth Cu(II) site in the prion protein

The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH(2) pentapeptide (P(5)) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(II) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO(3) to N(4) for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [Cu(II)(P(5))H(-2)] complex formed at pH 6.7 occurs at E (1/2)=0.04 V versus Ag/AgCl, whereas reversible oxidation of the [Cu(II)(P(5))H(-3)](-) complex formed at pH 10.0 occurs at E (1/2)=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794-6803, 2003) strongly suggests an N(3)O binding mode at physiological pH for the fifth Cu(II) site in the protein.     

The displacement of copper by iron at the specific binding sites of ovotransferrin

We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested.     

Structural study of the zinc and cadmium complexes of a type 2 plant (Quercus suber) metallothionein: insights by vibrational spectroscopy

Zn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT.     

Spectroscopic characterization of mononitrosyl complexes in heme--nonheme diiron centers within the myoglobin scaffold (Fe(B)Mbs): relevance to denitrifying NO reductase

Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.     

Computational studies of Cu(II)[peptide] binding motifs: Cu[HGGG] and Cu[HG] as models for Cu(II) binding to the prion protein octarepeat region

The binding of Cu(II) to the prion protein is investigated by computations at the B3LYP level of theory on models of the octarepeat domain of the prion protein. The models incorporate the functionality of the glycine (G) and histidine (H) residues which occur in the octarepeat domain, PHGGGWGQ. The copper complexes are designated Cu[HG] and Cu[HGGG]. Coordination to the metal via the imidazole ring of the histidine, the amide carbonyl groups, and the backbone nitrogen atom of the amide groups were examined, as well as several protonation/deprotonation states of each structure. EPR and CD titration experiments suggest that the octarepeat segments of the unstructured N-terminal domain of prion protein can bind Cu(II) in a 1:1 Cu-to-octarepeat ratio. The results identify the extent to which the Cu(II) facilitates peptide backbone deprotonation, and the propensity of binding in the forward (toward the C-terminus) direction from the anchoring histidine residue. A plausible mechanism is suggested for changing from amide O-atom to deprotonated amide N-atom coordination, and for assembly of the observed species in solutions of Cu[PrP] and truncated models of it. A structure is proposed which has the N2O2 coordination pattern for the minor component observed experimentally by EPR spectroscopy for the Cu[HGGG] model. The most stable neutral Cu[HGGG] structure found, with coordination environment N3O1, corresponds to that observed for Cu[HGGGW] and Cu[HGGG] both in the solid state and as the major component in solution at neutral pH.     

Spectroscopic and structural analyses of the copper(II) 2-D coordination polymer {[Cu2(BPP)4(NCS)4]}n (BPP = 1,3-bis(4-pyridyl)propane) comprising interpenetrated layers of (4, 4) topology

The synthesis and structural characterization of the 2-D Cu(II) coordination polymer namely {[Cu₂(BPP)₄(NCS)₄]}_n, where BPP is the nitrogen ligand 1,3-bis(4-pyridyl)propane, are described. Single crystal diffraction analysis shows that the asymmetric unit consists on two crystallographically independent Cu(II) ions that adopt a distorted octahedral geometry. Each Cu(II) center is coordinated by four nitrogen atoms from different BPP ligands and by other two nitrogen atoms from isothiocyanate groups. The BPP ligands link the metal centers generating an undulated two-dimensional net of (4, 4) topology. Two sets of two-dimensional sheets interlock each other in the same plane, giving rise to a twofold parallel interpenetrating network. EPR spectra indicate no magnetic coupling of the two individual Cu²⁺ centers.