COPPER-BINDING TO RABBIT LIVER METALLOTHIONEIN - FORMATION OF A CONTINUUM OF COPPER(I)-THIOLATE STOICHIOMETRIC SPECIES

Circular dichroism and ultraviolet absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(I)-thiolate binding clusters in rabbit liver metallothione in during addition of Cu+ to aqueous solutions of Zn-7-metallothionein 2 and Cd5Zn2-metallothionein 2. Mammalian metallothionein binds metal s in two binding sites, namely the alpha and beta domains. Spectral da ta which probe the distribution of Cu(I) between the two binding domai ns clearly show that both the site of binding (alpha or beta), and the structures of the specific metal-thiolate clusters formed, are depend ent on temperature and on the nature of the starting protein (either Z n-7-metallothionein or Cd5Zn2-metallothionein). CD spectra acquired du ring the addition of Cu+ to Zn-7-metallothionein show that Cu+ replace the bound Zn(II) in a domain-distributed manner with complete removal of the Zn(II) after addition of 12 Cu+. Spectral and metal analyses p rove that a series of Cu(I)-metallothionein species are formed by a no n-cooperative metal-binding mechanism with a continuum of Cu(I):metall othionein stoichiometries. Observation of a series of spectral saturat ion points signal the formation of distinct optically active Cu(I)-thi olate structures for the Cu9Zn2-metallothionein, Cu-12-metallothionein , and the Cu-15-metallothionein species. These data very clearly show that for Cu(I) binding to Zn-7-metallothionein, there are several key Cu(I) :metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu-12-metallothionein species ar e defined by bands located at 255(+) nm and 280(-) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data reco rded between 3 degrees C and 52 degrees C as Cu+ is added to Zn metall othionein show that copper-thiolate cluster formation is strongly temp erature dependent. These changes in spectral properties are interprete d in terms of kinetic versus thermodynamic control of the metal-bindin g pathways as Cu+ binds to the protein. At low temperatures (3 degrees C and 10 degrees C) the spectral data indicate a kinetically controll ed mechanism whereby an activation barrier inhibits formation of order ed copper-thiolate structures until formation of Cu-12-metallothionein . At higher temperatures (>30 degrees C) the activation barrier is ove rcome, allowing formation of new Cu(I)-thiolate clusters with unique s pectral properties, especially at the Cu9Zn2-metallothionein point. Th e CD spectra also show that a Cu-15-metallothionein species with a wel l-defined, three-dimensional structure forms at all temperatures, char acterized by a band near 335 nm, indicating the presence of digonal Cu (I). Complicated CD spectral changes are observed when Cu+ is added to Cd5Zn2-metallothionein. The spectral data are interpreted in terms of domain-distributed binding followed by rearrangement to form the doma in-specific product. In the Cu6Cd4- metallothionein species, the Cu+ a re ultimately bound specifically to the beta domain of the protein. Th is complex is characterised by the CD spectrum of the Cd4S'Cys'(11) in the alpha domain. The domain-specific product arises from the result of two interdependent driving forces, leading to formation of the Cu6S 'Cys'(9), beta-domain cluster and the Cd4S'Cys'(11) alpha-domain clust er. These findings imply physiological roles for the individual domain s of this protein. Further Cu+ addition yields the mixed metal Cu12Cd4 -metallothionein species which exhibits a unique CD spectrum with band s at 240, 268, 293 and 332 nm. Molecular modeling calculations were us ed to create a structure for the Cu-12-metallothionein 2 species, base d on domain stoichiometries identified by the spectroscopic data of Cu 6S'Cys'(11) (alpha domain) and Cu6S'Cys'(9) (beta domain). In accord w ith the CD spectral data, this structure involves exclusive trigonal c oordination of all 12 bound Cu+ to the 20 cysteinyl thiolates. All cys teinyl thiolates in the beta domain adopt bridging geometry, while cys teinyl thiolates in the alpha domain adopt both bridging and terminal geometries.