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Cu(I)‐ and proton‐binding properties of the first N‐terminal soluble domain of Bacillus subtilis CopA
Author(s) -
Zhou Liang,
Singleton Chloe,
Hecht Oliver,
Moore Geoffrey R.,
Le Brun Nick E.
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08422.x
Subject(s) - tetramer , dimer , chemistry , monomer , bacillus subtilis , linker , crystallography , chelation , copper , stereochemistry , biochemistry , biology , enzyme , inorganic chemistry , bacteria , organic chemistry , operating system , computer science , genetics , polymer
CopA, a P‐type ATPase transporter involved in copper detoxification in Bacillus subtilis , contains two soluble Atx1‐like domains separated by a short linker at its N‐terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans , 688–696]. To try to understand the contributions of each domain to the complex Cu(I)‐binding behaviour of this and related proteins, we purified a wild‐type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity ( K = ∼ 1 × 10 18 m −1 ) and underwent Cu(I)‐mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid–base properties revealed p K a values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct. Structured digital abstract• CopA and CopA bind by molecular sieving ( View interaction ) • CopA and CopA bind by cosedimentation in solution ( View interaction )