Mass spectrometric studies of Cu(I)-binding to the N-terminal domains of B-subtilis CopA and influence of bacillithiol

CopA is a Cu(I)-exporting transmembrane Pis-type ATPase from Bacillus subtilis. It contains two N-terminal cytoplasmic domains, CopAab, which bind Cu(I) with high affinity and to form higher-order complexes with multiple Cu(I) ions. To determine the precise nature of these species, electrospray ionisation mass spectrometry (ESI-MS) under non-denaturing conditions was employed. Up to 1 Cu per CopAab resulted in Cu coordination to one or both CopAab domains. At > 1 Cu/CopAab, two distinct dimeric charge state envelopes were observed, corresponding to distinct conformations, each with Cu-6(CopAab)(2) as its major form. The influence of the physiologically relevant low molecular weight thiol bacillithiol (BSH) on Cu(I)-binding to CopAab was assessed. Dimeric CopAab persisted in the presence of BSH, with previously undetected Cu-7(CopAab)(2) and Cu-6(CopAab)(2)(BSH) forms apparent.