Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal–protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.     

Spectroscopic identification of different types of copper centers generated in synthetic four-helix bundle proteins

Using a combined rational-combinatorial approach, stable copper binding sites were implemented in template-assembled synthetic four-helix bundle proteins constructed by three different helices with only 16 amino acid residues. These peptides include two histidines and one cysteine at positions appropriate for coordinating a copper ion. Sequence variations of the helices were made in the second coordination shell or even more remote from the copper binding site (i) to increase the overall stability of the metalloproteins and (ii) to fine-tune the structure and properties of the copper center. As a result, ca. 90% of the 180 proteins that were synthesized were capable to bind copper with a substantially higher specificity than those obtained in the first design cycle (Schnepf, R.; Horth, P.; Bill, E.; Wieghardt, K.; Hildebrandt, P.; Haehnel, W. J. Am. Chem. Soc. 2001, 123, 2186-2195). Furthermore, the stabilities of the copper protein complexes were increased by up to 2 orders of magnitude and thus allowed a UV-vis absorption, resonance Raman, electron paramagnetic resonance, and (magnetic) circular dichroism spectroscopic identification and characterization of three different types of copper binding sites. It could be shown that particularly steric perturbations in the vicinity of the His(2)Cys ligand set control the formation of either a tetragonal (type II) or a tetrahedral (type I) copper binding site. With the introduction of two methionine residues above the histidine ligands, a mixed-valent dinuclear copper binding site was generated with spectroscopic properties that are very similar to those of Cu(A) sites in natural proteins. The results of the present study demonstrate for the first time that structurally different metal binding sites can be formed and stabilized in four-helix bundle proteins.     

Intermolecular transfer of copper ions from the CopC protein of Pseudomonas syringae. Crystal structures of fully loaded Cu(I)Cu(II) forms

CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.     

Effects of thioether substituents on the O2 reactivity of beta-diketiminate-Cu(I) complexes: probing the role of the methionine ligand in copper monooxygenases

The activation of dioxygen by dopamine beta-monooxygenase (DbetaM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) is postulated to occur at a copper site ligated by two histidine imidazoles and a methionine thioether, which is unusual because such thioether ligation is not present in other O2-activating copper proteins. To assess the possible role of the thioether ligand in O2 activation by DbetaM and PHM, two new ligands comprising beta-diketiminates with thioether substituents were synthesized and Cu(I) and Cu(II) complexes were isolated. The Cu(II) compounds are monomeric and exhibit intramolecular thioether coordination. While the Cu(I) complexes exhibit a multinuclear topology in the solid state, variable-temperature 1H NMR studies implicate equilibria in solution, possibly including monomers with intramolecular thioether coordination that are structurally defined by DFT calculations. Low-temperature oxygenation of solutions of the Cu(I) complexes generates stable 1:1 Cu/O2 adducts, which on the basis of combined experimental and theoretical studies adopt side-on "eta(2)" structures with negligible Cu-thioether bonding and significant peroxo-Cu(III) character. In contrast to previously reported findings with related ligands lacking the thioether group, however (cf., Aboelella; et al. J. Am. Chem. Soc. 2004, 126, 16896), purging the solutions of the thioether-containing adducts with argon results in conversion to bis(mu-oxo)dicopper(III) species. A role for the thioether in promoting loss of O2 from the 1:1 Cu/O2 adduct and facilitating trapping of the resulting Cu(I) complex to yield the bis(mu-oxo) species is proposed, and the possible relevance of this role to that of the methionine in the active sites of DbetaM and PHM is discussed.     

CopC protein from Pseudomonas syringae: intermolecular transfer of copper from both the copper(I) and copper(II) sites

The CopC protein from Pseudomonas syringae pathovar tomato is expressed as one of four proteins encoded by the operon CopABCD that is responsible for copper resistance. It is a small soluble molecule (10.5 kDa) with a beta-barrel structure and features two distinct copper binding sites, which are highly specific for Cu(I) (K(D) > or = 10(-)(13)) and Cu(II) (K(D) approximately 10(-)(15)). These dissociation constants were estimated via ligand competition experiments monitored by electronic spectral and fluorescence probes. The chemistries of the two copper sites are interdependent. When the Cu(II) site is empty, the Cu(I) ion is oxidized by air, but when both sites are occupied, the molecule is stable in air. The availability of an unoccupied site of higher affinity induces intermolecular transfer of either Cu(I) or Cu(II) while maintaining free copper ion concentrations in solution at sub-picomolar levels. This intriguing copper chemistry is consistent with the proposed role of CopC as a copper carrier in the oxidizing periplasmic space. These properties would allow it to exchange either Cu(I) or Cu(II) with its putative partners CopA, CopB, and CopD, contrasting with the role of the Cu(I) (only) chaperones found in the reducing cytoplasm.     

Design and spectroscopic characterization of peptide models for the plastocyanin copper-binding loop

The Cu(II)- and Co(II)-binding properties of two peptides, designed on the basis of the active site sequence and structure of the blue copper protein plastocyanin, are explored. Peptide BCP-A, Ac-Trp-(Gly)(3)-Ser-Tyr-Cys-Ser-Pro-His-Gln-Gly-Ala-Gly-Met-(Gly )(3)-His-(Gly)(2)-Lys-CONH(2), conserves the Cu-binding loop of plastocyanin containing three of the four copper ligands and has a flexible (Gly)(3) linker to the second His ligand. Peptide BCP-B, Ac-Trp-(Gly)(3)-Cys-Gly-His-Gly-Val-Pro-Ser-His-Gly-Met-Gly-CONH(2), contains all four blue copper ligands, with two on either side of a beta-turn. Both peptides form 1:1 complexes with Cu(II) through His and Cys ligands. BCP-A, the ligand loop, binds to Cu(II) in a tetrahedrally distorted square plane with axial solvent ligation, while BCP-B-Cu(II) has no tetrahedral distortion in aqueous solution. In methanolic solution, distortion of the square plane is evident for both BCP-Cu(II) complexes. Tetrahedral Co(II) complexes are observed for both peptides in aqueous solution but with 4:2 peptide:Co(II) stoichiometries as estimated by ultracentrifugation. Cu(II) reduction potentials for the aqueous peptide-Cu(II) complexes were measured to be +75 +/- 30 mV vs NHE for BCP-A-Cu(II) and -10 +/- 20 mV vs NHE for BCP-B-Cu(II). The results indicate that the plastocyanin ligand loop can act as a metal-binding site with His and Cys ligands in the absence of the remainder of the folded protein but, by itself, cannot stabilize a type 1 copper site, emphasizing the role of the protein matrix in protecting the Cu binding site from solvent exposure and the Cys from oxidation.     

Spectroscopic studies of metal binding and metal selectivity in Bacillus subtilis BSco, a Homologue of the Yeast Mitochondrial Protein Sco1p

Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA-containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 A and by a weakly bound low-Z scatterer at 1.95 +/- 0.03 A. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 A and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CuA center of cytochrome c oxidase.     

Computer simulation of the interaction of Cu(I) with cys residues at the binding site of the yeast metallochaperone Cu(I)-Atx1

The copper binding site and electronic structure of the metallochaperone protein Atx1 were investigated using the combination of quantum mechanics methods and molecular mechanics methods in the ONIOM(QM:MM) scheme at the density functional theory (DFT) B3LYP/ 6-31G(d):AMBER level. The residues in the binding site, -Met13-Thr14-Cys15-Cu(I)-Cys18-Gly17-Ser16-, were modeled with QM and the rest of the residues with MM. Our results indicate that the structure for Cu(I)-Atx1 has the copper atom coordinated to two sulfur atoms from Cys15 (2.110 A) and Cys18 (2.141 A) with an angle S-Cu(I) -S of 166 degrees . The potential energy surface of the copper atom is used to estimate its binding energy and the force field for the copper ligands. The potential surface is shallow for the bending mode S-Cu-S, which explains the origin of the disorder observed in crystallographic and nuclear magnetic resonance studies. Using molecular dynamics for Cu(I)-Atx1 in a box of water molecules and in vacuum, with the force field derived in this work, we observed a correlated motion between the side chains of Thr14 and of Lys65 which enhances distortions in the S-Cu-S geometry. The results are compared with recent experiments and the previous models. The vibrational spectra for the copper ligands and for the residues in the binding site were computed. The localized modes for the copper ligands and the amide bands were assigned. The presence of the copper atom affects the amide bands' frequencies of the residues Cys15 and Cys18, giving resolved bands that can be used to sense changes in the binding site upon translocation of copper atom or interaction with target proteins. Furthermore, the EXAFS (extended X-ray absorption fine structure) spectrum of the proposed structure for Cu(I)-Atx1 was calculated and reproduced the experiments fairly well.     

Lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper-translocating ATPase (ATP7A), but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1 (15)N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased toward 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met, whereas at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.     

A Mets motif peptide found in copper transport proteins selectively binds Cu(I) with methionine-only coordination

Mets motifs, which refer to methionine-rich sequences found in the high-affinity copper transporter Ctr1, also appear in other proteins involved in copper trafficking and homeostasis, including other Ctrs as well as Pco and Cop proteins isolated from copper-resistant bacteria. To understand the coordination chemistry utilized by these proteins, we studied the copper binding properties of a peptide labeled Mets7-PcoC with the sequence Met-Thr-Gly-Met-Lys-Gly-Met-Ser. By comparing this sequence to a series of mutants containing noncoordinating norleucine in place of methionine, we confirm that all three methionine residues are involved in a thioether-only binding site that is selective for Cu(I). Two independent methods, one based on mass spectrometry and one based on rate differences for the copper-catalyzed oxidation of ascorbic acid, provide an effective K(D) of approximately 2.5 microM at pH 4.5 for the 1:1 complex of Mets7-PcoC with Cu(I). These results establish that a relatively simple peptide containing an MX(2)MX(2)M motif is sufficient to bind Cu(I) with an affinity that corresponds well with its proposed biological function of extracellular copper acquisition.     

Mechanistic studies of Cu(II) binding to amyloid-beta peptides and the fluorescence and redox behaviors of the resulting complexes

Due in large part to the lack of crystal structures of the amyloid-beta (Abeta) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal-Abeta complex has been difficult. In this work, we investigated the complexation of Cu(II) by Abeta through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Abeta can be reduced to Cu(I) using sodium borohydride and that both Abeta-Cu(II) and Abeta-Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Abeta fluorescence, commonly reported to be quenched upon Abeta-Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Abeta-Cu(II) complexes provides evidence that the sole tyrosine residue in Abeta is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to Abeta, the binding heterogeneity, and the influence of solution conditions on complex formation.     

Transfer of copper between bis(thiosemicarbazone) ligands and intracellular copper-binding proteins. insights into mechanisms of copper uptake and hypoxia selectivity

Bis(thiosemicarbazonato) complexes Cu(II)(Btsc) have attracted interest as promising metallodrugs and, in particular, as copper radiopharmaceuticals. Prototypes Cu(Atsm) and Cu(Gtsm) are membrane-permeable, but their metabolisms in cells are distinctly different: copper that is delivered by Cu(Gtsm) is trapped nonselectively in all cells, whereas copper that is delivered by Cu(Atsm) is retained selectively in hypoxic cells but is "washed out" readily in normal cells. We have studied copper-transfer reactions of these two complexes under various conditions, aiming to model their cellular chemistry. In Me2SO, both complexes exhibited reversible one-electron-reduction processes with Cu(Atsm) being more difficult to reduce than Cu(Gtsm) (E(1/2)'=-0.60 and -0.44 V, respectively, vs AgCl/Ag). Upon introduction of an aqueous buffer into Me2SO, the electrochemical reduction remained chemically reversible for Cu(Atsm) but became irreversible for Cu(Gtsm). However, the estimated difference in their reduction potentials did not change. Chromophoric ligand anions bicinchonate (Bca) and bathocuproine disulfonate (Bcs) were used as Cu(I) indicators to trace the destinations of copper in the reactions and to mimic cellular Cu(I)-binding components ("sinks"). While both BtscH2 ligands have high affinities for Cu(I) (KD in the picomolar range), they cannot compete with Cu(I) sinks such as the copper-binding proteins Atx1 and Ctr1c (or a mimic such as Bcs). In the presence of these proteins, reduction of Cu(II)(Btsc) leads to irreversible transfer of copper to the protein ligands. Endogenous reductants ascorbate and glutathione can reduce Cu(II)(Gtsm) in the presence of such protein ligands but cannot reduce Cu(II)(Atsm). These properties establish a strong correlation between the contrasting cellular retention properties of these complexes and their different reduction potentials. The endogenous reductants in normal cells appear to be able to reduce Cu(II)(Gtsm) but not Cu(II)(Atsm), allowing the latter to be washed out. The more reducing environment of hypoxic cells leads to reduction of Cu(II)(Atsm) and retention of its copper.     

Voltammetric measurement of copper(II)/organic interactions in estuarine waters

The voltammetry of copper in organic ligand/chloride media is dominated by the formation of CuCl^?₂ species and by induced adsorption of Cu(I) in organic coatings on the electrodes. These phenomena are utilised in a novel method for evaluating Cu(II)/organic ligand interactions, based on the principle of ligand exchange. The Cu(II)/organic species competes with glycine which forms copper glycinate. These two complexes can be distinguished voltammetrically: copper glycinate gives a higher surface excess of copper at a gelatin-coated hanging mercury drop electrode, partly because of the increased production of CuCl^?₂ from copper glycinate at the electrode surface. The method proved satisfactory for pure ligand/surfactant/chloride media and for estuarine waters. It is shown that there are two type of Cu(II)-binding ligand in estuarine waters: humic material (> 10^?6 mol l^?1, assuming 1:1 site binding) with polyelectrolyte-type binding, and discrete ligands (? 10^?6 M) with stability constants around 10⁹. The extent of Cu(II) binding by the humic material decreases down the estuary because of dilution and increased salinity.     

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.     

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.     

Galactose oxidase models: solution chemistry, and phenoxyl radical generation mediated by the copper status

Galactose oxidase (GO) is an enzyme that catalyzes two-electron oxidations. Its active site contains a copper atom coordinated to a tyrosyl radical, the biogenesis of which requires copper and dioxygen. We have recently studied the properties of electrochemically generated mononuclear Cu(II)-phenoxyl radical systems as model compounds of GO. We present here the solution chemistry of these ligands under various copper and dioxygen statuses: N(3)O ligands first chelate Cu(II), leading, in the presence of base, to [Cu(II)(ligand)(CH(3)CN)](+) complexes (ortho-tert-butylated ligands) or [(Cu(II))(2)(ligand)(2)](2+) complexes (ortho-methoxylated ligands). Excess copper(II) then oxidizes the complex to the corresponding mononuclear Cu(II)-phenoxyl radical species. N(2)O(2) tripodal ligands, in the presence of copper(II), afford directly a copper(II)-phenoxyl radical species. Addition of more than two molar equivalents of copper(II) affords a Cu(II)-bis(phenoxyl) diradical species. The donor set of the ligand directs the reaction towards comproportionation for ligands possessing an N(3)O donor set, while disproportionation is observed for ligands possessing an N(2)O(2) donor set. These results are discussed in the light of recent results concerning the self-processing of GO. A path involving copper(II) disproportionation is proposed for oxidation of the cross-linked tyrosinate of GO, supporting the fact that both copper(I) and copper(II) activate the enzyme.     

A strategy for the NMR characterization of type II copper(II) proteins: the case of the copper trafficking protein CopC from Pseudomonas Syringae

CopC from Pseudomonas syringae was found to be a protein capable of binding both Cu(I) and Cu(II) at two different sites. The solution structure of the apo protein is available, and structural information has been obtained on the Cu(I) bound form. We attempt here to set the limits for the determination of the solution structure of a Cu(II) protein, such as the Cu(II) bound form of CopC, in which the Cu(II) ion takes a type II coordination. The electron relaxation time is estimated from NMRD measurements to be 3 ns which leads to a correlation time for the nuclear spin-electron spin dipolar interaction of 2 ns. This information allowed us to tailor the NMR experiments and to fully exploit purely heteronuclear spectroscopy to assign as many signals as possible. In this way, 37 (13)C and 11 (15)N signals that completely escape detection with conventional approaches were assigned. Paramagnetic based structural constraints were obtained by measuring paramagnetic longitudinal relaxation enhancements (rho(para)) which allowed us to precisely locate the copper ion within the protein frame. Pseudocontact shifts (pcs's) were also used as constraints for 83 (1)H and 18 (13)C nuclei. With them, together with other standard structural constraints, a structure is obtained (and submitted to PDB) where information is only missing in a sphere with a 6 A radius from the copper ion. If we borrow information from EXAFS data, which show evidence of two copper coordinated histidines, then His 1 and His 91 are unambiguously identified as copper ligands. EXAFS data indicate two more light donor atoms (O/N) which could be from Asp 27 and Glu 89, whereas the NMRD data indicate the presence of a semicoordinated water molecule at 2.8 A (Cu-O distance) roughly orthogonal to the plane identified by the other four ligands. This represents the most extensively characterized structure of a type II Cu(II) protein obtained employing the most advanced NMR methods and with the aid of EXAFS data. The knowledge of the location of the Cu(II) in the protein is important for the copper transfer mechanism.     

Insights into the N-terminal Cu(II) and Cu(I) binding sites of the human copper transporter CTR1

Abstract

Copper transporter 1 (CTR1) is the main copper transporter in the eukaryotic system. CTR1 has several important roles: It binds Cu(II) ions that are present in the blood; it reduces those Cu(II) ions to Cu(I); and it subsequently transfers Cu(I) to the cytoplasmic domain, where the ion is delivered to various cellular pathways. Here, we seek to identify CTR1 binding sites for Cu(II) and Cu(I) and to shed light on the Cu(II)-to-Cu(I) reduction process. We focus on the first 14 amino acids of CTR1. This N-terminal segment is rich with histidine and methionine residues, which are known to bind Cu(II) and Cu(I), respectively; thus, this region has been suggested to have an important function in recruiting Cu(II) and reducing it to Cu(I). We utilize electron paramagnetic resonance (EPR) spectroscopy together with nuclear magnetic resonance (NMR) and UV-VIS spectroscopy and alanine substitution to reveal Cu(II) and Cu(I) binding sites in the focal 14-amino-acid segment. We show that H5 and H6 directly coordinate to Cu(II), whereas M7, M9, and M12 are involved in Cu(I) binding. This research is another step on the way to a complete understanding of the cellular copper regulation mechanism in humans.     

Computational studies of Cu(II)/Met and Cu(I)/Met binding motifs relevant for the chemistry of Alzheimer's disease

A systematic study of the binding motifs of Cu(II) and Cu(I) to a methionine model peptide, namely, N-formylmethioninamide 1, has been carried out by quantum chemical computations. Geometries of the coordination modes obtained at the B3LYP/6-31G(d) level of theory are discussed in the context of copper coordination by the peptide backbone and the S atom of a methionine residue in peptides with special emphasis on Met35 of the amyloid-beta peptide (Abeta) of Alzheimer's disease. The relative binding free energies in the gas phase, DeltaG(g), are calculated at the B3LYP/6-311+G(2df,2p)//B3LYP/6-31G(d) level of theory, and the solvation affects are included by means of the COSMO model to obtain the relative binding energies in solution, DeltaG(aq). A free energy of binding, DeltaG(aq) = -19.4 kJ mol(-1), relative to aqueous Cu(II) and the free peptide is found for the most stable Cu(II)/Met complex, 12. The most stable Cu(I)/Met complex, 23, is bound by -15.6 kJ mol(-1) relative to the separated species. The reduction potential relative to the standard hydrogen electrode is estimated to be E degrees (12/23) = 0.41 V. On the basis of these results, the participation of Met35 as a low affinity binding site of Cu(II) in Abeta, and its role in the redox chemistry underlying Alzheimer's disease is discussed.