The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

Abstract

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.

Nodule‐specific Cu+‐chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules

“…Within the symbiosome, Cu + would reach the bacteroid cytosol through yet‐to‐be‐determined transporters at the outer and inner membranes. There, Cu + ‐chaperone CopZ would transfer back the metal to Cu + ‐ATPase FixI (Kahn et al ., 1989; Patel et al ., 2014) that would be used, in turn, for cytochrome c oxidase metallation (Preisig et al ., 1996; Andrei et al ., 2021). However, in infected and/or noninfected cells MtNCC1 also transfers copper to other proteins such as the putative USP‐A identified, and likely a peroxiredoxin, and other proteins involved in plant–microbe interactions, or in SAM metabolism.…”

Copper management strategies in obligate bacterial symbionts: balancing cost and benefit

“…Copper is utilized by numerous enzymes involved in various essential metabolic pathways, such as electron transport [ 4 , 5 ], nitrogen metabolism [ 6 , 7 ], degradation of aromatic compounds [ 8 ], and oxygen-related reactions. It also plays a critical role in the cytochromes c oxidase of the respiratory chain [ 9 , 10 ], thus contributing to the generation of the proton motive force, the energy source of ATP synthase [ 11 ]. However, the evolutionary adoption of copper came with concomitant toxic metabolites.…”