Ternary complexes metal [Co(II), Ni(II), Cu(II) and Zn(II)]--ortho-iodohippurate (I-hip)--acyclovir. X-ray characterization of isostructural [(Co, Ni or Zn)(I-hip)(2)(ACV)(H(2)O)(3)] with stacking as a recognition factor

Four ternary metal--ortho-iodohippurate (I-hip)--acyclovir (ACV) complexes, [M(I-hip)(2)(ACV)(H(2)O)(3)] where M is Co(II) (1), Ni(II) (2), Cu (3) and Zn(II) have been obtained by reaction between the corresponding binary complexes M(II)(I-hip)(2)xnH(2)O and ACV. Three ternary complexes (M=Co, Ni and Zn) and the corresponding Zn(II)--ortho-iodohippurate binary derivative have been structurally characterized by X-ray diffraction: The studies show these three ternary complexes are isostructural and present, in solid state, an interesting stacking between the nucleobase and the aryl ring of the hippurate moiety, which probably promotes the formation of ternary complexes. Moreover, the two different ligands interact between them by means of ancillary hydrogen bonds with water molecules coordinated to the metal ion. It must be mentioned that these two recognition factors, hydrogen bonds plus stacking, could explain the reason for the isostructurality of these ternary derivatives with so different three metal ions, with diverses trends in coordination numbers and geometries. In solid state, there are two enantiomeric molecules that are related by an inversion center as the crystal-building unit (as a translational motif) for the ternary complexes.     

Solution 1H NMR investigation of Zn2+ and Cd2+ binding to amyloid-beta peptide (Abeta) of Alzheimer's disease

Elevated levels of zinc2+ and copper2+ are found chelated to the amyloid-beta-peptide (Abeta) in isolated senile plaque cores of Alzheimer's disease (AD) patients. However, the precise residues involved in Zn2+ ligation are yet to be established. We have used 1H NMR and CD to probe the binding of Zn2+ to Abeta(1-28). Zinc binding to Abeta causes a number of 1H NMR resonances to exhibit intermediate exchange broadening upon Zn2+ addition, signals in slow and fast exchange are also observed. In addition, there is a general loss of signal for all resonances with Zn2+ addition, suggestive of the formation of high molecular weight polymeric species. Perturbations in specific 1H NMR resonances between residues 6 and 14, and analysis of various Abeta analogues in which each of the three His residues have been replaced by alanine, indicates that His6, His13 and His14 residues are implicated in Zn-Abeta binding. Complementary studies with Cd2+ ions cause perturbations to 1H NMR spectra that are strikingly similar to that observed for Zn2+. Binding monitored at Val12 indicates a 1:1 stoichiometry with Abeta for both Zn2+ and Cd2+ ions. Circular Dichroism (CD) studies in the far-UV indicate quite minimal ordering of the main-chain with Zn2+ or Cd2+ addition. Changes in the far-UV are quite different from that obtained with Cu2+ additions indicating that Zn2+ coordination is distinct from that of Cu2+ ions. Taken together, these observations seem to suggest that Zn2+ coordination is dominated by inter-molecular coordination and the formation of polymeric species.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.     

Mechanistic studies of the astacin-like <Emphasis Type="Italic">Serratia</Emphasis> metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism

Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.     

Supramolecular structures of metronidazole and its copper(II), cobalt(II) and zinc(II) coordination compounds

In this work we present the synthesis and structural and spectroscopic characterization of Cu(II), Co(II) and Zn(II) coordination compounds with the antibiotic metronidazole ([double bond]emni). Coordination to metal ions is through its imidazolic nitrogen, while the hydroxyethyl and nitro groups act as supramolecular synthons. [Co(emni)(2)Br(2)], and [Zn(emni)(2)X(2)] (X(-)=Cl, Br) stabilize zig-zag chains, and a 2D supramolecular structure is formed by inter-chain contacts through inter-molecular hydrogen-bonding. Pleated sheet or layers are formed by [Co(emni)(2)Cl(2)] and [Cu(emni)(2)Cl(H(2)O)](2)Cl(2), respectively. The dinuclear Cu(II) compound [Cu(emni)mu(O(2)CMe)(2)](2) gives a one-dimensional zig-zag arrangement. The contribution of metal ions in metronidazole coordination compounds is shown in the stabilization of the different aggregate structures.     

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.     

Cyanide binding to Cu, Zn superoxide dismutase. An NMR study of the Cu(II), Co(II) derivative

The Cu,Co superoxide dismutase derivative, in which the native Zn(II) was replaced by Co(II), was investigated by 1H NMR spectroscopy at pH 7.0 in the presence of CN- and N-3. Addition of either anion produced large but remarkably different variations in the position of the histidine proton signals bound to the metal cluster. The resonances of the histidines bound to the copper broadened at low CN- concentrations (6 X10(-5)-16.5 X 10(-3) M KCN, in the presence of 1.5 mM protein) and narrowed again, with changed chemical shifts at [KCN] greater than 10(-2) M. At 7 degrees C two resonances split into two pairs of lines as a function of [CN-]. The temperature dependence of these resonances, in the presence of nonsaturating [CN-], suggests a slow exchange between two forms of the protein-bound copper in the presence of the anion. The apparent activation parameters associated with the interconversion of the two species indicate a local conformational change in the presence of CN-. No evidence of temperature dependence was seen in the spectrum in the presence of N-3, which, on the other hand, was fully removed from the copper by addition of CN-. No evidence was obtained for removal by CN- of a histidine bound to the copper as previously reported for low affinity anions at pH 5.5 (Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monanni, R., and Scozzafava, A. (1985) J. Am. Chem. Soc. 107, 4391-4396). These results indicate that CN- has a unique pattern of binding to the enzyme copper. Since catalytic and structural data indicate that CN- is the only appropriate substrate analogue for the Cu,Zn superoxide dismutase, data from anions with much less affinity may lead to misleading conclusions on the mechanism of anion and substrate binding to the enzyme.     

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.     

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.     

Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.     

Binding of zinc(II) and copper(II) to the full-length Alzheimer's amyloid-beta peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.     

Single-strand cleavage of DNA by Cu(II) and thiols: a powerful chemical DNA-cleaving system

Reduced lipoic acid, in the presence of cupric ions, introduced single-strand nicks into pSP64 plasmid DNA at micromolar concentrations, converting the supercoiled into open circular and, eventually, linear forms. The metal ion specificity of the reaction was investigated and, of Cu2+, Co2+, Cr3+, Fe3+, Fe2+, Ni2+, Mn2+ and Zn2+, only Cu2+ ions were catalysts for the thiol-induced DNA cleavage at these low concentrations. A wide range of thiols and dithiols was found to be active as DNA cleavers in the presence of Cu2+ ions.     

Adsorptive Removal and Recovery of U(VI), Cu(II), Zn(II), and Co(II) from Water and Industry Effluents

Tamarind fruit shell (TFS) was converted to a cation exchanger (PGTFS-SP-COOH) having a carboxylate functional group at the chain end by grafting poly(hydroxyethylmethacrylate) onto TFS (a lignocellulosic residue) using potassium peroxydisulfate-sodium thiosulfate redox initiator, and in the presence of N, N ′-methylenebisacrylamide as a cross-linking agent, followed by functionalization. The chemical modification was investigated using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and potentiometric titrations. The feasibility of PGTFS-SP-COOH for the removal of heavy metals such as U(VI), Cu(II), Zn(II), and Co(II) ions from aqueous solutions was investigated by batch process. The optimum pH range for the removal of meal ions was found to be 6.0. For all the metal ions, equilibrium was attained within 2 h. The kinetic and isotherm data, obtained at optimum pH value 6.0, could be fitted with pseudo-second-order equation and Sips isotherm model, respectively. The Sips maximum adsorption capacity for U(VI), Cu(II), Zn(II), and Co(II) ions at 30°C was found to be 100.79, 65.69, 65.97, and 58. 81 mg/g, respectively. Increase of ionic strength decreased the metal ion adsorption. Different wastewater samples were treated with PGTFS-SP-COOH to demonstrate its efficiency in removing metal ions from wastewater. The adsorbed metal ions on PGTFS-SP-COOH can be recovered by treating with 1.0 M NaCl + 0.5 M HCl for U(VI) ions and 0.2 M HCl for Cu(II), Co(II), and Zn(II) ions. Four adsorption/desorption cycles were performed without significant decrease in removal capacity. The results showed that PGTFS-SP-COOH developed in this study exhibited considerable adsorption potential for the removal of U(VI), Cu(II), Zn(II), and Co(II) ions from water and wastewaters.     

NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase.

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.     

The T to R transition in the copper(II)-substituted insulin hexamer. Anion complexes of the R-state species exhibiting type 1 and type 2 spectral characteristics.

The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.     

A room temperature magnetic susceptibility study on the cobalt derivatives of cuprozinc superoxide dismutase

A room temperature magnetic susceptibility study was carried out on several derivatives of Cu, Zn Superoxide dismutase: (1) E, Co(II) in which Co(II) binds to the normal Zn(II) site and the Cu(II) site is empty; (2) Co(II), Zn(II) in which Co(II) binds to the site normally occupied by Cu(II); (3) Co(II), Co(II) in which both the Zn(II) and the Cu(II) are replaced by cobalt; (4) Cu(II), Co(II) in which only the Zn(II) is replaced by cobalt. In this latter derivative the effect of the addition of increasing amounts of SCN⁻ on the magnetic susceptibility was also tested. For all the samples the magnetic susceptibility values resulted to be the sum of the contributions of the single paramagnetic ions and indicate that any magnetic coupling between the two metals is well below kT at room temperature.