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).     

Insight into the copper coordination environment in the prion protein through density functional theory calculations of EPR parameters

Density functional theory (DFT) calculations of Cu(II) electron paramagnetic resonance (EPR) parameters for the octarepeat unit of the prion protein were conducted. Model complexes were constructed and optimized using the crystal structure of the octarepeat unit of the prion protein. Copper g and A tensors and nitrogen hyperfine and quadrupole coupling constants were calculated using DFT. Solvent effects were incorporated using the conductor-like screening model as well as through the inclusion of explicit water molecules. Calculations using the model with an additional axial water molecule added to the coordination sphere of the Cu(II) metal center give the best qualitative agreement for the copper g and A tensors. The S-band experimental EPR spectra were interpreted in light of the DFT calculations of the directly coordinated nitrogen hyperfine coupling constants which indicate that the three directly coordinated nitrogen atoms in the octarepeat unit are not equivalent. These results demonstrate that DFT calculations of EPR parameters can provide important insight with respect to the structural interpretation of experimental EPR data. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH

The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu^II ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured “amyloidogenic” domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719] where we demonstrated that the physiologically relevant high affinity Cu^II coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that Cu^II is contained within a square planar (N/O)₃S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the Cu^II coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met → Ile “mutations” we show that the Cu^II coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one Cu^II(N/O)₃S center with the M(109) thioether coordinated to Cu^II and another Cu^II(N/O)₃S center with the M(112) thioether coordinated to Cu^II. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the Cu^II affinity of the peptide by two to seven fold, and renders the resulting Cu^II metallopeptides redox inactive. The biological implications of these findings are discussed.     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.     

A reassessment of copper(II) binding in the full-length prion protein

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.     

The conformation and orientation of copper(II)-bleomycin intercalated with DNA

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values ($\text{g}{}_{\mathrm{}}\text{=2.21}$, g_⊥ =2.04) and the hyperfine constant ($\text{A}{}_{\mathrm{}}\text{=175}\text{G}$) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75° with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhyperfine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside the double helix.     

Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.     

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrP^c) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrP^sc) leading to prion diseases. The first non-mammalian PrP^c was identified in chicken and it was found to keep many structural motifs present in mammalian PrP^c, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in June 2005 with corrections to the text. The author name LaMendola has been corrected to La Mendola.     

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.     

Cu(II) and Zn(II) ions alter the dynamics and distribution of Mn(II) in cultured chick glial cells

Previous studies revealed that Mn(II) is accumulated in cultured glial cells to concentrations far above those present in whole brain or in culture medium. The data indicated that Mn(II) moves across the plasma membrane into the cytoplasm by facilitated diffusion or counter-ion transport with Ca(II), then into mitochondria by active transport. The fact that 1–10 M Mn(II) ions activate brain glutamine synthetase makes important the regulation of Mn(II) transport in the CNS. Since Cu(II) and Zn(II) caused significant changes in the accumulation of Mn(II) by glia, the mechanisms by which these ions alter the uptake and efflux of Mn(II) ions has been investigated systematically under chemically defined conditions. The kinetics of [⁵⁴MN]-Mn(II) uptake and efflux were determined and compared under four different sets of conditions: no adducts, Cu(II) or Zn(II) added externally, and with cells preloaded with Cu(II) or Zn(II) in the presence and absence of external added metal ions. Zn(II) ions inhibit the initial velocity of Mn(II) uptake, increase total Mn(II) accumulated, but do not alter the rate or extent Mn(II) efflux. Cu(II) ions increase both the initial velocity and the net Mn(II) accumulated by glia, with little effect on rate or extent of Mn(II) efflux. These results predict that increases in Cu(II) or Zn(II) levels may also increase the steady-state levels of Mn(II) in the cytoplasmic fraction of glial cells, which may in turn alter the activity of Mn(II)-sensitive enzymes in this cell compartment.     

ESR and optical absorption studies on the copper(II) interaction with small peptides containing aromatic amino acids

The interaction of Cu(II) with di- and tripeptides each containing phenylalanine, tryptophan or histidine in the amino acid chain has been investigated by means of electron spin resonance (ESR) and optical absorption spectroscopy. Cu(II) complexes of dipeptides and tripeptides exhibit different magnetic and optical parameters. Dipeptide complexes have larger g -values and smaller {A –values than tripeptide complexes. When compared to dipeptide complexes, the d-d band of the central metal ion is blue shifted for tripeptide complexes. There are no significant differences in the behavior of Cu(II) peptide complexes containing phenylalanine or tryptophan. Complexes of histidine containing peptides, however, show modified spectra caused by the participation of the imidazole nitrogen in the coordination to Cu(II). The imidazole nitrogen seems to coordinate in-plane with other coordinating atoms or in an axial position depending on the kind of peptide.Part of the Ph.D. thesis of L.S., D-26Dedicated to Prof. Dr. H. Glubrecht on the occasion of his 60th birthday     

Synthesis, structure and properties of copper(II) and manganese(II) coordination polymers with 1,5-dinitronaphthalene-3,7-dicarboxylate

Two new metal-organic coordination polymers with 1,5-dinitronaphthalene-3,7-dicarboxylate (NNDC), [Cu₂(NNDC)₂(DMF)_1.8(DMSO)_2.2(H₂O)₂]·H₂O (1) and [Mn₃(NNDC)₃(DMSO)₄]·2DMSO (2) have been synthesized under solvothermal conditions and characterized by single crystal X-ray diffraction and Thermogravimetric Analysis (TGA). The structure of compound 1 consists of one-dimensional chains with copper ions being linked by the dicarboxylate ligands. The coordination chains are associated into ladder-like double chains through O-H?O hydrogen bonds and π-π interactions, and the ladders are packed in a cross fashion through further π-π interactions to give the three-dimensional structure. The Mn(II) compound exhibits a 3D framework with the pcu topology, in which [Mn₃(COO)₆] clusters as octahedral secondary building blocks are linked by the naphthalene spacers. Magnetic analyses were carried out based on both temperature- and field-dependent data, consistently suggesting relatively weak antiferromagnetic interactions within the carboxylate bridged [Mn₃(COO)₆] cluster.     

Design of histidine containing peptides for better understanding of their coordination mode toward copper(II) by CD spectroscopy

The systematic investigation of the copper(II) complexes of tripeptides Xaa-Xaa-His, Xaa-His-Xaa and His-Xaa-Xaa, where Xaa=Gly or Ala was performed by combined pH-metry, spectrophotometry, CD and in part EPR spectroscopy. The matrix rank analysis of the spectral data revealed the number of the coloured and optically active species as a basis for the solution speciation. A critical evaluation on the speciation and solution structure of the complexes formed is presented on the basis of their d-d band optical activity. The replacement of a Gly residue with the chiral Ala amino acid allowed us to gain decisive information on the solution structure of the complexes by CD spectroscopy. It was shown that the tripeptides with histidine in the third position formed CuH(-2)L species with (NH(2), 2N(-), ImN - where Im stands for imidazole) coordination sphere as a major species, and only the macrochelated CuL complexes as minor species around pH 5.0. In copper(II)-Xaa-His-Xaa tripeptide systems the CuH(-1)L (NH(2), N(-), ImN) is the most stable species at physiological pH, but the vacant fourth site around copper(II)ions is offered for further deprotonation, most probably resulting in mixed hydroxo species at low (<5 x 10(-4)M) metal ion concentrations, while a tetrameric complex is dominant when the copper concentration exceeds 3 x 10(-3)M. The histamine type coordination mode in CuL and CuL(2) complexes of His-Xaa-Xaa ligands predominates at low pH. The structural consequences drawn from the CD spectra for the mono and bis parent complexes were supported by theoretical calculations. CD spectra strongly suggest the participation of the imidazole nitrogen both in the Cu(2)H(-2)L(2) and CuH(-2)L complexes.     

A novel copper(II)-radical complex with ferromagnetic interaction: Synthesis, crystal structure and magnetic properties

A novel copper(II)-radical complex [Cu(NITmPy)(PDA)(H₂O)] · (H₂O) (1) (NITmPy = 2-(3′-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, H₂PDA = 2,6-pyridinedicarboxylic acid) has been synthesized and structurally characterized by X-ray diffraction methods. It crystallizes in the triclinic space group . The Cu(II) ion exists in a distorted square pyramid environment. The molecules of [Cu(NITmPy)(PDA)(H₂O)] · (H₂O) are connected as a two-dimensional structure by the intermolecular hydrogen bonds. Magnetic measurements show intramolecular ferromagnetic interactions between NITmPy and Cu(II) ion and intermolecular antiferromagnetic interactions in 1.     

Cytotoxic activity, X-ray crystal structures and spectroscopic characterization of cobalt(II), copper(II) and zinc(II) coordination compounds with 2-substituted benzimidazoles

Herein we present the synthesis, structural and spectroscopic characterization of coordination compounds of cobalt(II), copper(II) and zinc(II) with 2-methylbenzimidazole (2mbz), 2-phenylbenzimidazole (2phbz), 2-chlorobenzimidazole (2cbz), 2-benzimidazolecarbamate (2cmbz) and 2-guanidinobenzimidazole (2gbz). Their cytotoxic activity was evaluated using human cancer cell lines, PC3 (prostate), MCF-7 (breast), HCT-15 (colon), HeLa (cervic-uterine), SKLU-1 (lung) and U373 (glioblastoma), showing that the zinc(II) and copper(II) compounds [Zn(2mbz)₂Cl₂]·0.5H₂O, [Zn(2cmbz)₂Cl₂]·EtOH, [Cu(2cmbz)Br₂]·0.7H₂O and [Cu(2gbz)Br₂] had significant cytotoxic activity. The isostructural cobalt(II) complexes showed not significant activity. The cytotoxic activity is related to the presence of halides in the coordination sphere of the metal ion. Recuperation experiments with HeLa cells, showed that the cells recuperated after removing the copper(II) compounds and, on the contrary, the cells treated with the zinc(II) compounds did not. These results indicate that the mode of action of the coordination compounds is different.     

Immobilization of Cu(II) and Cr(IV) in basidiomycete-colonized sawdust

An investigation into the feasibility of removing Cu(II) and Cr(IV) from solution with basidiomycete (Gloeophylum sepiarium, Pleurotus sp.)-colonized sawdust was undertaken. Obeche (Triplochyton scleroxylon) sawdust exposed to the basidiomycetes for 1–3 months reduced the concentration of the metals in the solution to 22.0–84.4 mg/l. The supernatant from the centrifuged mixture of a solution of 100 mg metal ions/l and aqueous extract of a 3-month basidiomycete-degraded obeche sawdust contained lower concentration of the metal ions (38.6–75.4 mg/l). Unextracted sawdust of pigmented tropical timbers, African mahogany (Khaya ivorensis), black afara (Terminalia ivorensis) and camwood (Baphia nitida) exposed to the test basidiomycetes, removed Cu and Cr significantly better than the extracted sawdust. It is hypothesised that some products of basidiomycete wood-degradative activities were ligands which immobilized the test metals.     

A new porous 2D coordination polymer built by copper(II) and trimesic acid

A 2D porous material, Cu₃(tmen)₃(tma)₂(H₂O)₂ · 6.5H₂O [tmen = N,N,N′,N′-tetramethylethane-1,2-diamine; tmaH₃ = 1,3,5-benzenetricarboxylic acid/trimesic acid], has been synthesized and characterized by X-ray single crystal analysis, variable temperature magnetic measurements, IR spectra and XRPD pattern. The complex consists of 2D layers built by three crystallographically independent Cu(tmen) moieties bridged by tma anions. Of the three copper ions, Cu(1) and Cu(2) present distorted square pyramidal coordination geometry, while the third exhibits a severely distorted octahedral environment. The Cu(1)(tmen) and Cu(2)(tmen) building blocks bridged by tma anions give rise to chains with a zig-zag motif, which are cross-connected by Cu(3)(tmen)-tma polymers sharing metal ions Cu(2) through pendant tma carboxylates. The resulting 2D architecture extends in the crystallographic ab-plane. The adjacent sheets are embedded through the Cu(3)(tmen)-tma chains, leaving H₂O-filled channels. There are 6.5 lattice water molecules per formula unit, some of which are disordered. Upon heating, the lattice water molecules get eliminated without destroying the crystal morphology and the compound rehydrated reversibly on exposure to humid atmosphere. Magnetic data of the complex have been fitted considering isolated irregular Cu₃ triangles (three different J parameters) by applying the clumag program. The best fit indicates three close comparable J parameters and very weak antiferromagnetic interactions are operative between the metal centers.