Metallothionein: A diamagnetic metalthiolate cluster protein

Abstract

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.