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The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz. The protein part constitutes 53 amino acids. A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered. A family of 30 structures was generated. Root-mean-square deviation (rmsd) values from the average structure of 0.32 ± 0.13 A and 0.61 ± 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2–40. The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined. The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior. If the same metal–sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available. The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.

Iron(II)-substituted yeast metallothionein has been studied with Mossbauer spectroscopy. The iron in the protein is in the high-spin ferrous state. A maximum metal content of four iron(II)/molecule has been determined, with the four metal ions forming a diamagnetic cluster due to the antiferromagnetic exchange interaction between Fe2+ via bridging thiolates. In the case where the iron titration gives a value of less than four iron(II)/apoprotein, the metal ions are magnetically non-interacting, with each individual iron(II) behaving like iron(II) in reduced rubredoxin.

The formation of a ternary complex using cupric ions, basic amines of biological significance, and nucleotides was examined employing high-resolution nuclear magnetic resonance (NMR) with 13C and 31P and circular dichroism. The reaction of Cu2+ with each ligand was studied separately and as the ternary complex. From 13C-NMR data the assignment of the respective carbon atoms adjacent to a copper-binding atom was clearly shown. It is intriguing to note that even in such a ternary complex the N-7 of the purine ring, as well as the phosphate residue (measured by 13P-NMR spectroscopy) provide the preferred binding sites for the cupric ion. Marked interactions of the chelated cupric ion with the ribose moiety in the ternary complex were deduced from circular dichroic measurements. No such reactivity was seen when dAMP was employed. The existence of a ternary Cu · AMP · amine complex opens a number of new aspects regarding the reactivity of metal ions in the biochemistry of nucleic acids.

The initial binding of Cu2+ ot L-lysine, L-histidine, glycyl-histidine and histidyl-glycine in aqueous solutions was examined by 13C nuclear magnetic resonance spectroscopy. The measurements were carried out in a substantially improved way employing the pulse Fourier transform technique. Spectra of both high quality and resolution were obtained. Cu2+ complex formation with L-lysine occurred with the alpha-amino and carboxyl group attributable to the well expressed broadening effect of the 13C signals of the alpha-carbon atom and the carboxyl atom. The epsilon-amino group was not involved. Measurements of the Cu chelates using L-histidine and glycyl-histidine and histidyl-glycine confirmed the ambidentate nature of the histidine residue. It was concluded that an equilibrium exists between two Cu-complex species designated as histamine-like and histamine-like/glycine-like species. In the homogeneous histamine-like Cu complex, the Cu2+ is exclusively bound with 4 nitrogens, while in the other species one oxygen of the glycyl carboxyl group is involved in the Cu2+ binding. Blocking of this carboxyl groups by peptide bonding as found in histidyl-glycine favoured the formation of a Cu complex where the imidazole carbons of the histidyl residue were the most influenced species.

The preparation of pure metallothionein from Cd-pretreated rats and chicken was performed using short-time treatment with chloroform/ethanol followed by ion-exchange and gel chromatography. The protein contained 7 g-atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn-thionein remained homogeneous during Sephadex G-25, G-50 and G-75 gel filtration, during polyacrylamide disc-gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one-third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn-thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X-ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter- or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di-tert-butyloxy-carbonyl-l-cystine and tert-butyloxycarbonyl-l-cysteine as model compounds for high-molecular-weight and water-soluble cysteine compounds, respectively. From CD data it was concluded that Cd, Zn-thionein exists mainly as a random-coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X-ray photo-electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.

The intracellular distribution of 65Zn in rat liver has been studied in vivo and in vitro 1 The highest 65Zn uptake can be observed in the nuclear fraction. A maximum occurs after approx. 5 hours which is in contrast to the rapid rise of detectable 65Zn in the whole homogenate appearing immediately after the intraperitoneal injections of this metal ion. Further, 65Zn is retained by the nuclei for a much longer period. When Zn-deficient rats are employed 65Zn uptake is considerable enhanced at the beginning. However, the total 65Zn radioactivity of whole liver homogenate is only slightly increased. 2 The uptake of 65Zn by isolated liver nuclei suggests a strong temperature dependence in vitro. No 65Zn uptake at all can be measured when nuclei are employed which have been stored at –25° for two months. 3 An attempt has been made to isolate a double stranded DNA-65Zn complex from liver nuclei derived from 65Zn injected rats or from nuclei incubated with 65Zn. However, high ionic strength buffers or phenol displace 65Zn from the polynucleotide during the isolation procedure. 4 The intracellular 65Zn distribution in the liver portions remaining after partial hepatectomy in rats is in accordance with the results obtained above.