Spectroscopic and chemical approaches to the study of metal-thiolate clusters in metallothionein (MT).

Abstract

Structural properties of the metal-thiolate clusters in mammalian and vertebrate MTs were studied by employing various spectroscopic techniques. The stepwise probing of the metal-binding sites of the four-metal cluster domain (alpha-fragment) of rabbit MT-2 by Co(II) ions monitoring the V2[4A2----4T1(P)] ligand-field transition at 1260 nm and the EPR intensity of the high spin Co(II) complexes revealed spectral changes suggestive of a cluster structure with one metal-binding site of different symmetry. These results are in contrast with our previous findings on rabbit Co(II)7-MT-2 where a tetrahedral tetrathiolate Co(II) coordination at all stages of filling has been demonstrated. Similar titration studies on MT isolated from the crab Cancer pagurus revealed that all six metal-binding sites are bound in tetrahedral type of symmetry as documented by the development of the d-d absorption profile of crab Co(II)-MT as a function of increasing Co(II)/apoMT ratios. Evidence for clusters in the latter species is provided by the manifestation of magnetic exchange coupling in the EPR spectra when the last two Co(II) ions are bound. Unexpected spectral features in the d-d region of Co(II)6-MT and in the CD-profile of the Cd(II)6-MT seen after the addition of an excess of Co(II) or Cd(II), respectively, suggest the existence of a seventh metal-binding site of different symmetry in crab MT. Finally, the isolation of the metal-deficient rabbit Cd(II)6-MT-2 after exposure of the fully metal-occupied Cd(II)7-MT-2 to EDTA is reported. A combination of UV, CD and 113Cd NMR data obtained with both Cd(II)6- and Cd(II)7-MT-2 supports the occurrence of a labile metal-binding site in the three-metal cluster domain of mammalian MT.