Metal selectivity of clusters in rabbit liver metallothionein

Abstract

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i. e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism, 'I3Cd-NMR and EPR spectroscopy. In the case of Co/Cdmetallothionein, homometallic cluster occupation occurs, with the Cd(I1) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd-and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.Metallothioneins are members of a class of proteins, ubiquitous in nature, which are characterized by their unusual amino acid composition and structure. These proteins are rich in cysteine residues and bind with a high affinity d" metal ions such as Zn(II), Cd(II), Cu(1) and Hg(I1). Since the biosynthesis of metallothioneins is induced by these metals and by certain hormones, it is believed that they play a crucial role in the physiological handling of the essential trace metals zinc and copper, and in the detoxification of the non-essential metals cadmium and mercury [l, 21. Mammalian metallothioneins contain 61 or 62 amino acids out of which 20 are conserved cysteine residues involved in the binding of 7 divalent metal ions, i.e. Zn and/or Cd [3]. From spectroscopic and chemical studies, the presence of two adamantane-like metal-thiolate clusters of three and four metal ions tetrahedrally coordinated by thiolate ligands has been suggested [3 -81. The determination of the three-dimensional structure of metallothionein in crystals, by X-ray diffraction 191, and in solution, by two-dimensional NMR [lo -141, confirmed the spectroscopic cluster model. However, both three-dimensional models showed marked differences regarding the assignment of the sequence-specific cysteine-metal coordination bonds. The correctness of the NMR model has been confirmed by the recent reinvestigation of the crystal structure [ 151.Several lines of evidence suggest that the two clusters in metallothionein exhibit differential affinities for Cd, with the tighter binding occurring in the four-metal cluster [5, 16 -181. interestingly, although the Zn,-metallothionein form has been isolated from mammalian tissue, the highest cadmium content in metallothionein isolated from Cd-exposed animals was 5 mol Cd/mol protein [l], the remainder being Zn [19]. Moreover, in the native Znz,Cd5-metallothionein form the two met...

The building blocks of metallothioneins: heterometallic Zn2+ and Cd2+ clusters from first-principles calculations

“…This can be related to NMR data for the rabbit Cd 4 Zn 3 -MT, which revealed a non-uniform distribution of both metals, 68 and electrochemical studies that revealed several forms of human MT in solution depending on Cd 2+ and Zn 2+ concentration. 22 At moderate Cd 2+ load, the calculations reveal a mixture of many isomers.…”

“…Thus, no clusterspecific strain is apparent from the metal-ligand bond lengths; (5) for each stoichiometry of Zn 2+ and Cd 2+ , all possible isomers have been characterized and ranked according to relative free energy and the most stable identified; (6) no correlation between stability and angular structural distortion of the sites is observed; (7) specific sites display substantial metal ion selectivity: site 380 in cluster A selectively prefers Cd 2+ , with other sites being more or less neutral to Zn 2+ /Cd 2+ differentiation. In cluster B, site 378 has larger selectivity for Cd 2+ , and 376 has larger selectivity for Zn 2+ ; (8) the most stable distribution in clusters at low Cd 2+ concentration is computed to be Zn 4 68 We have identified which individual isomers occur. The driving force for this preference is enthalpic and can be explained from electrostatic consideration of the thiolate positions.…”

“…Evidence of several forms of heteroclusters of Cd 4 Zn 3 -MT stoichiometry has been obtained from 113 Cd NMR studies. 68 This suggests that the heteroclusters in MTs consist of more isomers in thermodynamic equilibrium. NMR studies found that the Zn 2+ ions in the B cluster are more labile than those in the A cluster under exposure to nitric oxide.…”

A two‐dimensional NMR study of Co(II)7 rabbit liver metallothionein

“…In the present study, the native Zn(I1) metal ions have been replaced with the paramagnetic Co(I1) ions with the aim to learn more about the structure of the zinc protein. Indeed, Co(I1) is an excellent biochemical probe for Zn(1I) [28]; upon Co(I1) to Zn(I1) replacement, only minor changes (= 5%) in cluster volume are to be expected [29].…”

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

“…in order to probe for metal selectivity of MT, we have spectroscopically characterized the cluster forms generated upon binding of various combinations of two different divalent metal ions to metalfree metallothionein, i.e. Co(II), Cd(II), Zn(I1) and Fe(I1) [19]. These studies revealed that homometallic cluster occupation occurs in the case of various ratios of Co/Cd-MT and Fe/Cd-MT.…”