title: Formation of mammalian Cu8-metallothionein in vitro: evidence for the existence of two Cu(I)4-thiolate clusters.
abstract: 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 Cu12-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 Cu4-thiolate clusters [Freedman, J. H., Powers, L., & 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 (Cu8-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 Cu8-MT species between 12 and 14 cysteines take part in Cu(I) coordination.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biochemisches Institut der Universität Zürich, Switzerland.
authors.forename: D L
authors.initials: DL
authors.lastname: Pountney
authors.forename: I
authors.initials: I
authors.lastname: Schauwecker
authors.forename: J
authors.initials: J
authors.lastname: Zarn
authors.forename: M
authors.initials: M
authors.lastname: Vasák
mesh_terms: D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements; D008280:Magnetics; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D013438:Sulfhydryl Compounds; C007121:copper thionein; D003300:Copper; D008668:Metallothionein