Copper Binding to Rabbit Liver Metallothionein

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Abstract
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 metallothionein during addition of Cu+ to aqueous solutions of Zn7‐metallothionein 2 and Cd5Zn2‐metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the α and β domains. Spectral data which probe the distribution of Cu(I) between the two binding domains clearly show that both the site of binding (α or β), and the structures of the specific meta‐thiolate clusters formed, are dependent on temperature and on the nature of the starting protein (either Zn7‐metallothionein or Cd5Zn2‐metallothionein). CD spectra acquired during the addition of Cu+ to Zn7‐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 prove that a series of Cu(I)‐metallothionein species are formed by a non‐cooperative metal‐binding mechanism with a continuum of Cu(I):metallothionein stoichionietries. Observation of a series of spectral saturation points signal the formation of distinct optically active Cu(I)‐thiolate structures for the Cu9Zn2‐metallothionein, Cu12‐metallothionein, and the Cu15‐metallothionein species. These data very clearly show that for Cu(I) binding to Zn7‐metallothionein, there are several key Cu(I):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu12‐metallothionein species are defined by bands located at 255(+) nm and 280(‐) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data recorded between 3°C and 52°C as Cu+ is added to Zn‐metallothionein show that copper‐thiolate cluster formation is strongly temperature dependent. These changes in spectral properties are interpreted in terms of kinetic versus thermodynamic control of the metal‐binding pathways as Cu+ binds to the protein. At low temperatures (3°C and 10°C) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper‐thiolate structures until formation of Cu12‐metallothionein. At higher temperatures (> 30°C) the activation barrier is overcome, allowing formation of new Cu(I)‐thiolate clusters with unique spectral properties, especially at the Cu9Zn2‐metallothionein point. The CD spectra also show that a Cu15‐metallothionein species with a well‐defined, three‐dimensional structure forms at all temperatures, characterized 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 domain‐specific product. In the Cu6Cd4‐metallothionein species, the Cu+ are ultimately bound specifically to the β domain of the protein. This complex is characterised by the CD spectrum of the Cd4S′Cys′11 in the α domain. The domain‐specific product arises from the result of two interdependent driving forces, leading to formation of the Cu6S′Cys′9, β‐domain cluster and the Cd4S′Cys′11α‐domain cluster. These findings imply physiological roles for the individual domains of this protein. Further Cu+ addition yields the mixed metal Cu12Cd4‐metallothionein species which exhibits a unique CD spectrum with bands at 240, 268, 293 and 332 nm. Molecular modeling calculations were used to create a structure for the Cu12‐metallothionein 2 species, based on domain stoichiometries identified by the spectroscopic data of Cu6S′Cys′11, (α domain) and Cu6S′Cys′9 (β domain). In accord with the CD spectral data, this structure involves exclusive trigonal coordination of all 12 bound Cu+ to the 20 cysteinyl thiolates. All cysteinyl thiolates in the β domain adopt bridging geometry, while cysteinyl thiolates in the α domain adopt both bridging and terminal geometries.

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