Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu1+ to the less toxic Cu2+. Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb3-type cytochrome oxidase (cbb3-Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb3-Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF.

Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation.

Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extra-cellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that co-translational export of the protein CutF regulates translation of the down-stream cutO-encoded multi-copper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is co-translationally exported by the signal recognition particle pathway. Binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its co-translational export via its C-terminal ribosome stalling-like motif. This allows the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO, which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and often, are located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using co-translational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation.