Structure of the metal clusters in rabbit liver metallothionein

Abstract

Cadmium-113 nuclear magnetic resonance ("13Cd NMR) has been used to determine the structures of the multiple cadmium binding sites in the two major isoproteins of rabbit liver metallothionein. The isotopically "13Cd-labeled metallothionein used in these studies was isolated from the livers of rabbits that had been subjected to repeated injections of "13CdCl2. The native protein isolated from these livers contains an appreciable amount of Zn in addition to Cd, ranging from 2-3 mol per mol of protein out of a total metal content of 7 mol per mol of protein. The "13Cd NMR spectrum of Cd, Zncontaining metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixture of species containing different relative amounts of Zn and Cd. Replacement of the native Zn with "13Cd in vitro gave a protein whose "13Cd NMR spectrum was much simpler, containing eight distinct multiplets with chemical shifts ranging from 611-670 ppm. The origin of the multiplet structures has been shown to be "13Cd-''3Cd scalar cou Fing arising from two-bond interactions between "13Cd ions linked to one another by bridging cysteine thiolate ligands. The size and structures of the metal clusters in the protein were determined by the application of selective homonuclear "13Cd decou ling techniques. Analysis of these data showed that rabbit iver metallothionein contains two separate metal clusters, one containing four Cd2+ ions and the other containing three. These two clusters, whose structures are the same in both isoproteins, have been designated "cluster A" and "cluster B," respectively. Structures for the clusters are proposed that account for the "13Cd spin coupling data and the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of eight multiplets rather than the seven that would be expected on the basis of the number of metal binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.Metallothioneins are a ubiquitous class of sulfhydryl-rich, low molecular weight proteins that are believed to play a central role in metal metabolism. Since the initial discovery in 1957 of the cadmium-and zinc-containing proteins from equine kidney cortex (1), metallothioneins have been characterized from a variety of other mammals (including man) and from such widely divergent sources as birds, fish, microorganisms, and invertebrates (2, 3). The widespread occurrence of metallothionein in nature suggests that it serves an important biological function, though its exact role has yet to be defined. Most attention has centered on the hypothesis that the protein acts as a heavy metal detoxifying agent by sequestering cadmium, mercury, and other harmful metal ions. Bolstering this suggestion is the observation that exposure of many organisms ...

Interactions of Bismuth Complexes with Metallothionein(II)

“…The seven metal ions are present in clusters of four and three metals bound to bridging and terminal cysteine thiolate ligands, with metal-tothiolate ratios of M 4 S 11 and M 3 S 9 for the ␣-and ␤-domains, respectively (23). When both Zn 2ϩ and Cd 2ϩ are present, Cd 2ϩ binds preferentially to the ␣-domain, whereas Zn 2ϩ is found preferentially in the ␤-domain (23,28). A Zn 2 Cd three-metal cluster (␤ domain) in MT has the same structure as a Cd 3 cluster (23).…”

Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

“…Thus, as shown in Fig. 6 its Cd content is reflected by a '13Cd NMR spectrum with five distinct and two overlapping Cd resonances with chemical shifts between 605 ppm and 695 ppm a region characteristic of Cd-thiolate clusters (Otvos and Armitage, 1980;Coleman, 1993).…”

Modulation of nitric oxide‐mediated metal release from metallothionein by the redox state of glutathione in vitro

“…The high amount of cysteine residues in MTs (30% of all amino acids are cysteine) allows these proteins to coordinate multiple mono (Cu + , Ag + ) or divalent metals (Zn 2+ , Cd 2+ ). Mammalian MTs bind seven divalent metals in two separate domains [4]. Three metals are bound in an M 3 Cys 9 cluster in the N-terminal b-domain, while an M 4 Cys 11 four metal cluster is formed in the C-terminal a-domain [4].…”

“…Therefore, we looked for a possible regulatory mechanism for the interplay between NO production and metal release from MTs in order to understand this dual expression of opposing proteins. As the metal is held in place by thiolate ligands [4] in MTs, other thiols may well influence the metal-bond formation and breakage. The major low-molecular mass thiol compound in plants and animals is the tripeptide L-c-glutamyl-L-cysteinyl-glycine also known as glutathione (GSH in reduced form and GSSG in its oxidized form).…”

“…Mammalian MTs bind seven divalent metals in two separate domains [4]. Three metals are bound in an M 3 Cys 9 cluster in the N-terminal b-domain, while an M 4 Cys 11 four metal cluster is formed in the C-terminal a-domain [4]. Of the four known mammalian MT isoforms [2], the two best studied and most widely occurring isoforms (1 and 2) are most abundant in parenchymatous tissues, i.e.…”