Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The metallothioneins (MT) are a class of low molecular weight, cysteine rich proteins found in almost all living things. The high sulfhydryl content imparts to these proteins the unique ability to bind a wide range of metals. The metallothioneins have been implicated in the storage and transport of essential metals, such as zinc and copper, and in the detoxification of toxic metals, such as cadmium. However, the exact physiological functions of these proteins remain a matter of speculation. In particular, little is known about the interactions of copper with MT. In this thesis, the structural and mechanistic properties of copper binding to metallothionein is investigated using the spectroscopic techniques of circular dichroism, luminescence, and to a lesser extent, UV-visible absorption spectroscopy. These spectroscopic results are interpreted in terms of the Cu(I):MT stoichiometries at which distinct spectral characteristics are observed, the coordination geometry of the bound Cu(I) atoms, and the energetics of the binding reaction.;The binding of Cu(I) to rabbit liver Zn-MT showed that a continuum of Cu(I)-MT species are formed, with distinct spectroscopic markers for the {dollar}\rm Cu\sb9Zn\sb2{dollar}-MT, {dollar}\rm Cu\sb{lcub}12{rcub}{dollar}-MT, and {dollar}\rm Cu\sb{lcub}15{rcub}{dollar}-MT species. These unique spectral characteristics indicate that these species represent transition points in the Cu(I)-thiolate structural motifs. The use of Cd(II) as a probe of the remaining tetrahedral binding sites after Cu(I) addition to Zn-MT indicates that Cu(I) binding occurs in a domain-distributed manner.;The fine regulation between the kinetically-favoured and the thermodynamically-favoured binding pathways is also examined for Cu(I) binding to rabbit liver MT. The thermodynamic reaction pathway, which results in formation of the domain specific {dollar}\rm Cu\sb6Scys\sb9\ \beta{dollar} domain cluster, is favoured by increasing the temperature of the reaction, by using Cd-MT rather than Zn-MT as the initial protein species, by adding a competitive thiolate ligand, and by using the demetallated apo-MT to bind the Cu(I). These results are interpreted in terms of their possible physiological significance.;A structural model of the significant {dollar}\rm Cu\sb{lcub}12{rcub}{dollar}-MT species is proposed based on the spectroscopic data of Cu(I) binding to Zn-MT. This structure fulfills the physical constraints of the peptide, as well as chemical constraints of synthetic Cu(I)-thiolate complexes. This model is used to gain a clearer insight into the binding properties of copper-metallothioneins and the further reactions which this species may undergo.;The spectroscopic characterization of Cu(I) binding to metallothionein is applied to an in vivo investigation of the role of metallothionein in copper metabolism. The intracellular formation of copper-metallothionein is observed within the yeast Saccharomyces cerevisiae. An examination of the luminescence of whole yeast cells grown in a copper-containing medium indicates that metallothionein plays a major role in the overall mechanism of copper uptake, storage, and enzymatic incorporation within the cell.



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