Clusters In Metallothionein Research Articles

Study on model clusters of metallothionein: Structure and its kinetics of zinc transfer reaction to apo-carbonic anhydrase

In order to investigate the relationship between the structure and functions of metallothioneins, [(CH 3) 4N] 2[Zn 4(SC 6H 5) 10], as a model cluster of metallothionein has been synthesized. The structure of the model cluster has been examined by a CAD-4 x-ray diffractometer and the kinetics of zinc transfer reaction to apocarbonic anhydrase has been studied. Structural analysis and kinetic study show that compared with the model cluster the clusters in metallothionein are structurally more loose and they have a similar reactivity of zinc transfer.

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features.

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., Kägi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.

Spectroscopic and chemical approaches to the study of metal-thiolate clusters in metallothionein (MT).

Structural properties of the metal-thiolate clusters in mammalian and vertebrate MTs were studied by employing various spectroscopic techniques. The stepwise probing of the metal-binding sites of the four-metal cluster domain (alpha-fragment) of rabbit MT-2 by Co(II) ions monitoring the V2[4A2----4T1(P)] ligand-field transition at 1260 nm and the EPR intensity of the high spin Co(II) complexes revealed spectral changes suggestive of a cluster structure with one metal-binding site of different symmetry. These results are in contrast with our previous findings on rabbit Co(II)7-MT-2 where a tetrahedral tetrathiolate Co(II) coordination at all stages of filling has been demonstrated. Similar titration studies on MT isolated from the crab Cancer pagurus revealed that all six metal-binding sites are bound in tetrahedral type of symmetry as documented by the development of the d-d absorption profile of crab Co(II)-MT as a function of increasing Co(II)/apoMT ratios. Evidence for clusters in the latter species is provided by the manifestation of magnetic exchange coupling in the EPR spectra when the last two Co(II) ions are bound. Unexpected spectral features in the d-d region of Co(II)6-MT and in the CD-profile of the Cd(II)6-MT seen after the addition of an excess of Co(II) or Cd(II), respectively, suggest the existence of a seventh metal-binding site of different symmetry in crab MT. Finally, the isolation of the metal-deficient rabbit Cd(II)6-MT-2 after exposure of the fully metal-occupied Cd(II)7-MT-2 to EDTA is reported. A combination of UV, CD and 113Cd NMR data obtained with both Cd(II)6- and Cd(II)7-MT-2 supports the occurrence of a labile metal-binding site in the three-metal cluster domain of mammalian MT.

Interaction of lysine residues with the metal thiolate clusters in metallothionein.

Metallothioneins are unique diamagnetic metal thiolate cluster proteins. Both vertebrate and invertebrate forms contain, besides their large cysteine content (30%), up to 14% lysine plus arginine. In the amino acid sequences, the basic residues are juxtaposed to cysteine residues and have been suggested to play a role in neutralizing the excess negative charge of the metal thiolate complexes [Kojima, Y., Berger, C., Vallee, B. L., & Kägi, J. H. R. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3413-3417]. To document such a function, we compared the susceptibility of the lysine residues in cadmium and zinc metallothioneins and in the metal-free S-carboxamidomethyl derivative toward arylation by trinitrobenzenesulfonic acid. The results show an at least 20-fold lower initial rate of reaction with the metal-containing as opposed to that with the metal-free form, indicating a protective effect of metal complex formation on the lysine residues, the degree of protection being dependent on the nature of the metal. The modification of the lysine residues by trinitrobenzenesulfonic acid produces changes in the CD spectral features of the cadmium thiolate cluster structure. The lowered chemical reactivity of the lysine residues in the metal-containing form correlates with an upward displacement of their average pKa's to 10.9 from 10.3 in the metal-free S-carboxamidomethyl derivative. We attribute these effects at least in part to hydrogen bonding of the positively charged epsilon-amino groups to the thiolate ligands of the negatively charged cadmium thiolate units.

Spectroscopic evidence for metal-thiolate clusters in complexes of Cd(II) with dithiol hexapeptides and in Cd-Metallothionein

Metallothioneins constitute a novel family of widely occurring metal-thiolate proteins which are engaged in the metabolism and the detoxification of posttransitional metal ions (Zn(II), Cd(II), Cu(I), etc.). All mammalian forms characterized are single chain proteins of 61 amino acids residues with binding sites for 7 metal ions [1]. The large metal-binding capacity is conditioned by the presence of 20 cysteine residues which provide thiolate ligands for the formation of two adamantane-like metal-thiolate cluster structures containing 3 and 4 metal ions, respectively [2,3]. The unique occurrence of these clusters in metallothionein is believed to be related to the presence of 7-Cys-X-Cys-sequences in the polypeptide chain where X stands for an amino acid residue other than Cys. To explore the metal complexing features of such dithiol sequences, we have now chemically synthesized by the Merrifield procedure the hexapeptides Ser-Cys-Val-Cys-Ala-Ala, Ala-Cys-Lys-Cys-Ala-Ala and Ala-Cys-Ser-Cys-Ala-Ala and have examined the spectroscopic features of their complexes with Cd(L) and related metal ions. Stepwise addition of Cd(II) to aqueous solutions of these peptides yields complexes of 1:2, 2:3 and 1:1 metal-to-peptide stoichiometry (Fig. 1(a)). The first two complexes display features ▪ typical of tetrahedral tetrathiolate coordination also seen in Cd(II)-metallothionein [4] (Fig. 1(c)). The 2:3 complex is thought to be a binuclear cluster complex composed of two tetrahedral cadmium-thiolate units connected via two bridging thiolate ligands. It differs from the mononuclear 1:2 complex by a slight red shift of the absorption envelope which can be attributed to the greater polarization of the bridging sulfur ligands by the metal. In addition, its formation is accompanied by a nearly complete loss of the of the strong circular dichroism denoting the greater symmetry its structure. The spectral shift signalling the formation of the ligand-bridged binuclear complex is most clearly indicated by the emergence of a maximum near 260 nm in the difference-difference absorption spectrum (Fig. 1(b)). Spectral changes entirely analogous to those accompanying the successive formation of the mononuclear and binuclear tetrahedral Cd(II)-peptide complex also occur in the course of reconstituting Cd(II)-metallothionein from Cd(II) and apometallothionein (Figs. 1(c) and (d)). Under the conditions employed (pH 8), Cd(II) binds at first to separate tetrathiolate sites. However, with all thiolate ligands becoming occupied, a red shift develops signifying the change-over to the clustered structure containing 40% bridging ligands. Interestingly, these spectral changes are much less pronounced when Cd(II) is incorporated at lower pH. Below pH, the binding of successive equivalents of Cd(II) is, in fact, accompanied by a blue shift of the Cd(II)-thiolate absorption envelope indicating an initial preferential formation of thiolate-bridged structures. Hence, it would appear that depending on pH the building-up of the metal-thiolate clusters in metallothionein proceeds through different pathways.

Metallothionein: A diamagnetic metalthiolate cluster protein

Metallothionein (MT) is an ubiquitously occurring, extremely sulfur- and metal-rich protein (mol. wt 6–7000) which plays a role in the metabolism and the detoxification of several essential and nonessential trace metals. All mammalian forms consist of a single polypeptide chain with a total of 61 amino acid residues of which 20 are cysteines. Each molecule binds 7 bivalent metal ions, most commonly Zn(II) and Cd(II). All cysteine residues participate in metal coordination through formation of metalmercaptide bonds. Their position in the polypeptide chain is preserved in all mammalian forms. Unique features are the CysXCys sequences (where X stands for an amino acid other than Cys) occurring 7 times along the chain. MT is an elongated nonglobular protein with an axial ratio of about 6 [1, 2]. As documented by 1H NMR and IR spectroscopy, it is a compact molecule with some secondary structure; however, it has not been crystallized as yet [3]. Hence, detailed structural information comes exclusively from spectroscopic studies. The electronic absorption spectrum is dominated by contributions from the electron transfer transitions of the metalthiolate complexes superimposed upon the plain absorption spectrum of the polypeptide chain. The positions of the first electron transfer band are strongly metal-dependent. The locations displayed in the derivatives of MT containing Zn, Cd, Hg or Pb are in accordance with Jørgensen's optical electronegativity scale. They coincide very closely with the shifts predicted for tetrahedral tetrathiolate model complexes, thus suggesting the same metal coordination in MT [4]. This is corroborated by the spectroscopic properties of MT reconstituted with Co(II). The visible and near-IR absorption spectrum of the green-colored derivative shows, besides the Co(II)thiolate electron transfer bands, dd maxima at 600, 682 and 743 nm belonging to the ν 3[ 4A 2 → 4T 1(P)] transition and at 1230 nm belonging to the ν 2[ 4A 2 → 4T 1(F)] transition. These are diagnostic of distorted tetrahedral tetrathiolate high-spin Co(II) complexes, a conclusion further substantiated by the magnetic circular dichroism (MCD) and electron spin resonance (ESR) spectra [5]. Independent evidence for the tetrahedral structure is also available for Zn(II)MT based on extended X-ray absorption fine structure (EXAFS) measurements [6] and for Cd(II)MT based on perturbed angular correlation of gamma ray (PAC) ▪ measurements using MT which contains the shortlived excited isomer 111mCd(II) [7]. The involvement of four sulfur ligands in the coordination of each metal ion is reconcilable with the overall Me(II) 7(Cys −) 20 stoichiometry of the molecule only if the metal complexes are sharing some thiolate ligands, thereby forming metal-thiolate clusters. This conjecture is now also confirmed experimentally. Thus the partitioning of the metal-coordinating ligands into bridging and non-bridging (terminal) thiolate ligands is manifested in a substantial broadening of the sulfur core electron binding energy profile in the X-ray photoelectron spectrum (ESCA) of MT as compared to those of elemental sulfur or monothiols [3]. Direct proof for clustering of the metal centers comes from the demonstration of metalmetal interactions by magnetic resonance spectroscopy. Thus in 113Cd-enriched MT 113Cd NMR resonances are split into multiplets by 113Cd 113Cd scalar coupling [8] and in fully substituted Co(II)MT the ESR-resonances are largely suppressed due to antiferromagnetic spin-canceling. This latter effect is also confirmed by magnetic susceptibility measurements [9]. By monitoring the changes in ESR amplitude and in magnetic susceptibility attending the stepwise incorporation of Co(II) into the protein, it is moreover possible to follow the building-up of the cluster structure. This process is biphasic. Up to binding of 4 equivalents of Co(II) the paramagnetic signals increase as expected for magnetically noninteracting high-spin complexes. However, upon further addition of Co(II), these features are lost again, signalling the transition to the magnetically interacting oligonuclear structures [9]. This two-step mode of metal-binding in which separate Co(II) tetrathiolate complexes are formed first is compatible with the model of two separate metal-thiolate clusters in MT deduced for 113Cd NMR homonuclear decoupling experiments [10] and from proteolytic cleavages studies [11] as well as from a 113Cd NMR study on cluster formation in progress in this laboratory. However, the actual steric organization of these clusters remains to be determined. An attractive structure consistent with all spectroscopic information at hand is the cage-like adamantane decahedron built up of thiolate units of tetrahedral symmetry (Fig. 1). The remarkable simplicity of the spectra and the unique capacity of MT to accommodate stoichiometrically different paramagnetic and diamagnetic metal ions, i.e., Ni(II), Co(II), Zn(II), Cd(II), Hg(II), Pb(II) and Bi(III), and to force them into environments of tetrahedral microsymmetry could be optimally accounted for by such a model or a variation of it. The need for these regular bioinorganic structures in MT would explain the remarkable preservation of the positions of all cysteine residues throughout mammalian evolution.