Analogous copper(I) coordination in metallothionein from yeast and the separate domains of the mammalian protein

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Abstract
The three-dimensional structures of both vertebrate Cu12-metallothionein (class 1) and yeast Cu8-thionein (class 2) are still unknown. The different copper:protein stoichiometry compared with that of the (ZnCd)7-metallothioneins was expected to alter the metal-thiolate cluster structure considerably. In order to avoid possible domain interactions in the hepatic rat metallothionein, separate chemically synthesized α-and β-domains were used rather than the apoprotein. Apo yeast thionein, and the α-and β-domains of rat liver metallothionein-2 were reconstituted by Cu(I) titration. Reconstitution steps were monitored using spectroscopic methods including luminescence emission and circular dichroism. Upon UV irradiation a linear increase in intensity of the orange-red luminescence was observed near 600 nm up to 6 Cu eq using either compound regardless of the different cysteine sulfur content (yeast thionein 12S, α-domain 11S, β-domain 9S). The characteristic dichroic properties of the yeast copper-protein between 240 and 400 nm were in good agreement with those of the respective class 1 metallothionein domains. All observed Cotton bands were of similar shape and appeared in the same wavelength regions. However, the molar ellipticities were less pronounced in the α-and β-fragments employed. There appears to be a striking similarity between the oligonuclear Cu(I) binding centers in all metallothionein species.

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