Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition

In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.     

Secondary ion mass spectrometry of nonvolatile isocyanide complexes of silver(I) and copper(I)

A series of silver and copper coordination complexes has been studied using secondary ion mass spectrometry (SIMS). Results are presented for the monomeric silver(I) complexes [Ag(CNR)₄]X, where R = cyclohexyl for X  ClO₄⁻, and R = methyl or t-butyl for X  PF₆⁻. Likewise, Cu(I) complexes [Cu(CNR)₄]PF₆, where R =methyl, t-butyl, or cyclohexyl, were examined. The presence of AgL₂⁺ (L represents the intact RNC ligand) and the absence of AgL₃⁺ and AgL₄⁺ species attests to the gas phase stability of two-coordinate silver(I). Similar results to these were obtained for the Cu(I) complexes, with the exception of [Cu(CNCH₃)₄]PF₆ whose spectrum contains CuL₄⁺, CuL₃⁺, CuL₂⁺, CuL⁺, and Cu⁺ ions. The latter result reflects the enhanced stability of the tetrahedral Cu(I) geometry compared to Ag(I) in the gas phase. Cross labeling experiments and isotopic labeling studies have provided insights into fragmentation mechanisms. Ligand exchange occurs when mixtures are examined. These exchange reactions provide evidence for extensive molecular mixing which can accompany SIMS even under low primary ion dose conditions. Cluster ion formation as well as the observation of α-cleavage of the NC bonds of RNC ligands have been observed and these results are discussed. Granulated graphite and ammonium chloride were employed to study matrix effects. Granulated graphite enhanced NC cleavage for the silver complexes but had little effect on the relative abundance of silver cluster ions. On the other hand, copper cluster ions were more sensitive to matrix effects.     

Biochemical characterization of CopA,the Escherichia coli Cu(I)-translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal β-strand from the β-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into Cu(I) transfer processes. The adaptable nature of the Cu(I) coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.     

Glutathione-mediated transfer of copper(I) into American lobster apohemocyanin.

Copper in the cytosol of the hepatopancreas of the American lobster, Homarus americanus, occurs as copper-metallothionein [Cu(I)-MT] and as a copper-glutathione complex [Cu(I)-GSH]. The latter can act in vitro as the source of Cu(I) in the reconstitution of lobster apohemocyanin, whereas Cu(I)-MT cannot. Here we report on the mechanism of the GSH-mediated reconstitution. Binding of Cu(I) to apohemocyanin was measured by its effect on the protein's fluorescence, by ultrafiltration experiments and size-exclusion HPLC. Reconstitution of CO and O2 binding was studied using the [Cu(I)...Cu(I)-CO] fluorescence of hemocyanin and its Cu-O2-Cu charge-transfer band as spectral probes. The hemocyanin oligomer has 1 (1.02 +/- 0.09) high-affinity (apparent Kdiss = 1.67 +/- 0.40 microM) external binding site for ionic Cu(I) per subunit. Binding of Cu(I) to this site is fast and reversible and is followed by a slow, irreversible incorporation of copper into the protein matrix. Movement of the first copper through the matrix to the active site is the rate-limiting step in the reconstitution process. Mononuclear copper sites, once formed, are rapidly converted into biologically active, binuclear copper sites. In accordance with this reaction sequence, the restoration of CO/O2 binding by hemocyanin is a first-order reaction with a half-time of 100 +/- 5 min at pH 6.0. Reconstitution is extremely pH-dependent and proceeds best at those pH values where the architecture of the copper pocket of hemocyanin is open as judged from its extremely low affinity for oxygen and its very fast oxygen dissociation rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cu-metallothioneins (Cu(I)8-MTs) in LEC rat livers 13 weeks after birth still act as antioxidants

Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long-Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)8-MTs). Titration of Cu(I)8-MTs with CuCl2 indicates that Cu(I)8-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)8-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)8-MTs with superoxide and hydroxyl radicals were estimated to be 2 x 10(6) and > or = 10(12) M(-1)s(-1), respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)8-MTs from LEC rat livers just before hepatitis still act as antioxidants.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-aliphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine, and glutathione have been investigated, with the goal of understanding the relationship between RS- -Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto amino acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]0), pH, and the structure of the redox partners. With excess cysteine present at 25 degrees C, pH 5.0, I = 0.2 M (NaOAc), decay of 1:1 cys-S- -Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)2+(6.1 X 10(3) M-1 sec-1), Cu(Me5dien)2+ (2.7 X 10(3) M-1 sec-1), Cu(en)22+ (2.1 X 10(3) M-1 sec-1), and Cu(dien)22+ (4.7 X 10(3) M-1 sec-1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results. Moderate reactivity decreases in the cysteine-Cu(dien)2+, Cu(Me5dien)2+ reactions with increasing [H+] (pH 4-6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS- -Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)2+ and Cu(Me5dien)2+. This behavior suggests the intermediacy of di-mu-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)2+ and Cu(Me5dien)2+ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me6tren)2+ (k(25 degrees C) 7.5 X 10(7) M-2 sec-1, I = 0.2M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR- -forming pathway. Formation of a coordinated disulfide in the rate-determining step is proposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me6tren)Cu(I) product.     

M-S vibrational study in three-coordinate thiolato compounds (NEt4)2[M(SC6H4-p-X)3] and (NEt4)

By using p-substituted benzenethiolate ligands, the novel three-coordinate copper(I) and silver(I) thiolato complexes (NEt4)2[Cu(SC6H4-p-X)3] (X=Cl (1) and Br (2)), (NEt4)2[Ag(SC6H4-p-X)3] (X=Cl (3) and Br (4)) and novel clusters (NEt4)2[M4(mu-SC6H4-p-Cl)6] (M=Cu (5) and Ag(6)) have been prepared and structurally characterized by single crystal X-ray diffraction. All the complexes have three-coordinate sites having point-group D3h symmetry. The three-coordinate mononuclear silver(I) complexes 3 and 4 are the first examples. The M-S stretching bands were determined by far-IR and FT-Raman spectroscopies; nu(Cu-S) 363-372 cm(-1) and nu(Ag-S) 353-363 cm(-1). These results indicate that M-S stretching vibration energy in the three-coordinate metal(I) site of the mononuclear compounds or clusters is around 340-380 cm(-1), and it is a useful tool for determining their coordination modes.     

Convenient preparation of [CuX(PCy3)2](X = Br, I, SCN, N3) complexes. X-ray crystal structure of [Cu(N3)(PCy3)2] (Cy = cyclo-C6H11)

The labile cations [Cu(F-BF₃)(PCy₃)₂] and [Cu(OTf)(PCy₃)₂] are versatile precursors for the formation of [Cu(X)(PCy₃)₂] (X = Br, I, SCN, N₃) complexes by metathesis with NaX. The azide [Cu(N₃)(PCy₃)₂] is triclinic, space group , a = 9.755(4), B = 22.78(1), C = 9.284(6) Å, = 96.76(3), β = 115.36(3), γ = 94.20(5)°, Z = 2.     

New copper(I) phosphane complexes of dihydridobis(3-nitro-1,2,4-triazolyl)borate ligand showing cytotoxic activity

New copper(I) complexes of the type [H(2)B(tz(NO2))(2)]Cu[PR(3)](2) (1-5), [H(2)B (tz(NO2))(2)]Cu[dppe] (6) and [H(2)B(tz(NO2))(2)]Cu[PR(3)] (7, 8) have been synthesized from the reaction of CuCl, potassium dihydrobis(3-nitro-1,2,4-triazol-1-yl)borate, K[H(2)B (tz(NO2))(2)], and mono- or bi-dentate tertiary phosphanes. The complexes obtained have been characterized by elemental analyses and FT-IR in the solid state, and by NMR ((1)H and (31)P{(1)H}) spectroscopy in solution. Selected complexes 1, 3 and 5 have also been tested against a panel of several human tumor cell lines in order to evaluate their cytotoxic activity. Complexes 1 and 5 showed IC(50) values appreciably lower than those exhibited by cisplatin, the most used metal-based antitumor drug. It is worth noting that all three tested Cu(I) complexes appear to be particularly effective against A549 carcinoma cells that are resistant to cisplatin treatment.     

Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA

CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser(46)-containing beta3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV-visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

Copper(I) complexes of penicillamine and glutathione

Equilibrium analysis of a model system for the in vivo reactions between penicillamine and Cu(I), the penicillamine-glutathione-Cu(I) system, indicates that in a certain concentration range the use of penicillamine as a drug will not disturb the normal Cu(I) metabolism. The equilibrium data required for this analysis were obtained by emf titrations on the Cu(I)-glutathione (H3A) and the Cu(I)-pencillamine (H2A) systems at 25 degrees C. in 0.5 M NaClO4 medium, using glass and copper amalgam electrodes; the data were analyzed first by various graphical methods and then by a general least squares computer program. The results show that mononuclear Cu(I) species Cu(HA)2 form in both systems with stability constants log beta 122 of 38.8 (glutathione) and 39.18 (penicillamine); in addition, the polynuclear Cu5A43- species forms in the penicillamine system and the mononuclear CuHA- species might form in the glutathione system. The results are discussed in relation to the therapeutic use of penicillamine as well as in relation to the toxic action of copper on living cells.     

Structural study of copper(I)-bleomycin

Previous NMR studies on Cu(I)-bleomycin have suggested that this adduct has a geometry distinct from Fe(II)BLM. The coordination chemistry of this bleomycin derivative has been investigated through the extension of the NMR data reported previously, and the use of molecular dynamics calculations. The data collected from the NMR experiments support the coordination to the metal center of the primary and secondary amines in beta-aminoalanine and the pyrimidine ring. The detection in the NMR spectra of the signal derived from the amide hydrogen in beta-hydroxyhistidine indicates that this amide is protonated in Cu(I)-bleomycin, precluding participation of the pyrimidinyl carboxamide nitrogen in the coordination of Cu(I), as previously reported. Three-dimensional solution structures compatible with the NMR data have been assayed for Cu(I)-bleomycin for the first time by way of molecular dynamics calculations, and two models showing four and five coordination have been found to be those that better fit the experimental data. In both models the primary amine in beta-aminoalanine is coordinated such that it is located on the same side, with respect to the coordination cage, as the peptide linker fragment. This result seems important for the favored models to be compatible with either their possible oxidation to become one of the reported structures for Cu(II)BLM, or their transformation into Fe(II) adducts able to cause DNA damage.     

Cu(I) binding properties of a designed metalloprotein

The Cu(I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an α-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu(I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu(I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600 nm emission. It was further found that the exogenous reductant tris(2-carboxyethyl)phosphine (TCEP) can competitively inhibit the binding of Cu(I) to the low affinity site of the peptide, but does not interact with Cu(I) clusters.     

Coordination of three and four Cu(I) to the alpha- and beta-domain of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.