FORMATION OF MAMMALIAN CU-8-METALLOTHIONEIN IN-VITRO - EVIDENCE FOR THE EXISTENCE OF 2 CU(I)(4)-THIOLATE CLUSTERS

Copper accumulates in metallothionein (Cu-MT) in copper overload diseases, such as Wilson's disease and Bedlington Terriers disease. The in vitro formation of the Cu-12-MT form comprising two Cu(I)(6)(CysS)(9,11) cores is well documented. However, lysosomal Cu-MT isolated from canine liver contains 8 Cu(I) ions in two proposed adamantane-like Cu-4-thiolate clusters [Freedman, J. H., Powers, L., and Peisach, J. (1986) Biochemistry 25, 2342]. The present studies have been carried out in an effort to learn more about the Cu(I)-thiolate cluster species formed upon the sequential incorporation of Cu(I) ions into metal-free MT from rabbit liver. On the basis of changes in the electronic absorption, circular dichroism (CD), magnetic circular dichroism (MCD), and luminescence spectra, besides the formation of a molecular species with 12 Cu(I) equivalents, evidence for the existence of a distinct MT complex with 8 Cu(I) equivalents (Cu-8-MT) was obtained. Analysis of the metal-dependent absorption envelope of Cu(I)-MT between 240 and 360 nm permitted the discrimination between predominantly CysS-Cu(I) charge-transfer (LMCT) (240-260 nm) and cluster-localized Cu(I) (d-s) transitions (260-360 nm). Accordingly, the decrease in the ratio of intensities of LMCT to d-s bands from 2.6 to 2.4 on going from 8 to 12 Cu(I) equivalents was attributed to the formation of Cu-MT species with different cysteine ligand to metal stoichiometries. The results suggest that while in CU12-MT all 20 thiolate ligands participate in metal binding, in the Cu-8-MT species between 12 and 14 cysteines take part in Cu(I) coordination. The low-temperature luminescence spectrum (77 K) of Cu-8-MT is characterized by low- and high-energy emission bands at 610 nm (tau similar to 130 mu s) and 425 nm (tau similar to 50 mu s), respectively, In contrast, the corresponding spectrum of the two Cu-6 clusters in Cu-12-MT exhibits only the low-energy band at 610 nm (tau similar to 130 mu s). The measured lifetimes (tau) are consistent with emissions from triplet excited states assigned to mixed d-s/LMCT in origin. Similar luminescence behavior has been observed with crystallographically defined inorganic Cu(I)6 and CU(I)(4) cluster models where it has been attributed to a shorter Cu...Cu separation in the latter case [Ford, P. C., and Vogler, A. (1993) Acc. Chem. Res. 26, 220]. Accordingly, the occurrence of the high-energy band in Cu-8-MT provides evidence for the existence of Cu(I)(4) clusters in this species. On the basis of these data, it is concluded that the Cu-8-MT species contains two CU(I)(4)(CysS)(6-7) clusters and is thus analogous to that isolated from canine liver (Bedlington Terriers disease). It is suggested that the formation of distinct Cu-8- and Cu-12-MT species reflects the relative concentrations of copper and protein present in solution.