The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb₃ oxidase assembly

Abstract

Summary Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu‐detoxification and Cu delivery for cytochrome c oxidase (cbb3‐Cox) assembly depend on two distinct Cu‐exporting P1B‐type ATPases. The low‐affinity CopA is suggested to export excess Cu and the high‐affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb3‐Cox biogenesis. In most organisms, CopA‐like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI‐like ATPases is unknown. Here we identified a CopZ‐like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox‐sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu‐sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb3‐Cox activity is reduced, suggesting that CopZ not only supplies Cu to P1B‐type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI‐CopZ protein complex in native R. capsulatus membranes.

Cu Homeostasis in Bacteria: The Ins and Outs

“…CopZ from the Gram-positive bacterium E. hirae was the first characterized bacterial Cu-chaperone and shown to be required for copper resistance and copper export [ 170 , 171 ]. In bacteria, CopZ genes are often, but not always [ 172 ], encoded in one operon together with the Cu-exporting P 1B -type ATPase CopA and Cu-sensing transcriptional regulators [ 173 , 174 ]. In E. coli , a CopZ chaperone is synthesized upon ribosomal frame-shifting during translation of the copA mRNA [ 175 ].…”

“…Cu(I)-binding to CopZ has been shown to induce dimerization [ 181 , 182 ], in which Cu(I) is present as a tetra-nuclear Cu complex in a Cu 4 (CopZ) 2 stoichiometry [ 182 ]. The physiological role of CopZ dimers is not entirely clear and dimerization might only occur at high Cu concentrations [ 172 , 183 ]. Furthermore, dimerization is inhibited by glutathione, and high cellular glutathione concentrations possibly prevent CopZ dimerization in vivo [ 145 , 184 ].…”

“…The proteobacterium R. capsulatus contains only one copZ , which is required for both Cu resistance and full assembly of cbb 3 -type Cox [ 172 ]. This dual role of CopZ is supported by the observation that it can be detected in a complex with either CcoI or CopA in R. capsulatus membranes [ 172 ].…”

“…The proteobacterium R. capsulatus contains only one copZ , which is required for both Cu resistance and full assembly of cbb 3 -type Cox [ 172 ]. This dual role of CopZ is supported by the observation that it can be detected in a complex with either CcoI or CopA in R. capsulatus membranes [ 172 ]. Thus, CopZ supplies Cu not only to CopA-dependent detoxification but also to cuproprotein biogenesis via the alternative Cu(I)-exporting ATPase CcoI, dedicated to cbb 3 -Cox assembly [ 172 ].…”

“…N-MBS are also present in CopA2-like ATPases, which bind Cu(I) with high affinity, but have a low turn-over rate. However, the role of the N-MBS in these ATPases has not been studied so far, although interactions of R. capsulatus CcoI with CopZ were shown [ 172 ], but whether this involves the N-MBS domain remains to be defined. For the eukaryotic ATP7B, it was also shown that Cu(I) binding influences the structure of the N-terminus, which contains six N-MBS [ 243 ].…”