Open AccessStructure of the metal clusters in rabbit liver metallothionein
Author(s) -
James D. Otvos,
Ian M. Armitage
Publication year - 1980
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.77.12.7094
Subject(s) - metallothionein , homonuclear molecule , cadmium , chemistry , metal , cysteine , metal ions in aqueous solution , metalloprotein , crystallography , stereochemistry , molecule , biochemistry , organic chemistry , enzyme
Cadmium-113 nuclear magnetic resonance (113 Cd NMR) has been used to determine the structures of the multiple cadmium binding sites in the two major isoproteins of rabbit liver metallothionein. The isotopically113 Cd-labeled metallothionein used in these studies was isolated from the livers of rabbits that had been subjected to repeated injections of113 CdCl2 . 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. The113 Cd NMR spectrum of Cd, Zn-containing 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 with113 Cdin vitro gave a protein whose113 Cd 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 be113 Cd-113 Cd scalar coupling arising from two-bond interactions between113 Cd 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 homonuclear113 Cd decoupling techniques. Analysis of these data showed that rabbit liver 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 the113 Cd 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 the113 Cd-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.