Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

Liang Zhou, Chloe Singleton, Oliver Hecht, Geoffrey R. Moore, Nick E. Le Brun

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6 Citations (Scopus)

Abstract

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 × 1018 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 pKa 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.
Original languageEnglish
Pages (from-to)285-298
Number of pages14
JournalFEBS Journal
Volume279
Issue number2
Early online date12 Nov 2011
DOIs
Publication statusPublished - Jan 2012

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