Comparative analysis of metal binding characteristics of copper chaperone proteins, Atx1 and ATOX1.

The metal binding properties of the human copper chaperone ATOXI and its yeast homologue Atxl have been characterized. Complexes of these proteins with Cu(I), Ag (1), Cd(II) and Hg(II) were studied by native gel electrophoresis, chemical cross-linking followed by SDS-PAGE, as well as by size exclusion chromatography, mutagenesis and UV-visible absorption spectroscopy. Results indicate that binding of different metals to either ATOXI or Atxl altered conformation of subunit structure and the oligomerization state of the proteins. Furthermore, it has been demonstrated that freshly reduced apoprotein is capable to convert Cu(ll) to Cu(l) stoichiometrically to the amount of protein present, while oxidized protein is only twenty per cent as active. Titration of Cu(ll) with either oxidized or reduced protein resulted in similar increase in absorbance at 254 nm, implicating Cu-thiolate formation in both forms of the protein, but titration with Ag(i) caused the increase in absorbance at 254 nm with the reduced protein only. These data indicate that Cu(1), Ag(1), Hg(ll) and Cd(ll) are all capable of binding to ATOXI and Atxl, but the characteristics of the binding to these copper chaperones differ for different metals.