The N-terminal soluble domains of Bacillus subtilis CopA exhibit a high affinity and capacity for Cu(I) ions.
CopA from Bacillus subtilis is a Cu(I)-transporting P-type ATPase involved in resistance to high levels of environmental copper. At its N-terminus are two soluble domains, a and b, that, when generated in isolation from the membrane part, have previously been shown to exhibit unusual Cu(I)-binding behaviour: at >1 Cu(I) per CopAab the protein dimerises, resulting in the formation of a species with luminescence properties characteristic of a solvent-shielded Cu(I) cluster. Further insight into the Cu(I)-binding properties of CopAab are now reported. We demonstrate that the initial binding of Cu(I) occurs with very high affinity (K = -4 x 10(17) M(-1)) and that CopAab can accommodate up to 4 Cu(I) per protein and remains dimeric at higher Cu(I)-loadings. Fitting of UV-visible, near UV CD, fluorescence and luminescence spectroscopic titration data supports a model in which Cu(I) binds sequentially to CopAab, and also provides estimates of the association constants for Cu(I)-binding and dimerisation steps. Finally, low molecular weight thiols are shown not to affect the initial binding of Cu(I), but significantly influence binding at levels >1 Cu(I) per CopAab such that dimerisation is inhibited, though not abolished.
Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, UK.
Chloe
C
Singleton
Nick E
NE
Le Brun
D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D008970:Molecular Weight; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry
D001426:Bacterial Proteins; C071263:CopA protein, Bacteria; D013438:Sulfhydryl Compounds; D003300:Copper