C-Terminal Domain of the Membrane Copper Transporter Ctr1 from Saccharomyces cerevisiae Binds Four Cu(I) Ions as a Cuprous-Thiolate Polynuclear Cluster: Sub-femtomolar Cu(I) Affinity of Three Proteins Involved in Copper Trafficking

Abstract

The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a CuI4(μ-S-Cys)6 cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant KD = (K1K2K3K 4)1/4 = 10-19 for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (β2 = 1019.8). Equivalent experiments for the yeast chaperone At..