Metallothionlin (MT) domains of different origins,exhibiting distinct, highly conserved cysteine positions, show differences in metal-cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 beta domains, was chosen as a basic model to study the structure-function relationship among the clusters. The possible influence of the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster beta (c) and beta (N) domains, each having nine cysteines and binding three M2+ ions, and a modified domain beta (C-N), in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD,Cd-113 NMR, and H-1 NMR spectroscopies. The synthetic native domains (Cd(3)beta (c) and Cd(3)beta (N)) displayed spectroscopic properties, metal-binding affinities, and kinetic reactivity similar to those of the hole protein. In contrast, the modified Cd(3)beta (C-N) domain was unusually reactive and, in the presence of Chelax, a metal-ion chelating resin, was converted to a Cd-5(beta (C-N))(2) dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd(3)beta (N) toward lobster Cd(3)beta (C-->N) dr in mammalian MT1 or MT2 toward Cd(3)beta -MT3 (GIF) can destabilize their structures.