AUTHOR=Andrei Andreea , Di Renzo Maria Agostina , Öztürk Yavuz , Meisner Alexandra , Daum Noel , Frank Fabian , Rauch Juna , Daldal Fevzi , Andrade Susana L. A. , Koch Hans-Georg 

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

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.712465

DOI=10.3389/fmicb.2021.712465

ISSN=1664-302X

ABSTRACT=<p>Copper (Cu)-transporting P<sub>1B</sub>-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P<sub>1B</sub>-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 <italic>cbb</italic><sub>3</sub>-type cytochrome oxidase (<italic>cbb</italic><sub>3</sub>-Cox) in different species. Here, we reconstituted purified CcoI of <italic>Rhodobacter capsulatus</italic> 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 <italic>cbb</italic><sub>3</sub>-Cox. CcoI and <italic>cbb</italic><sub>3</sub>-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 <italic>in vivo</italic> and sufficient to provide Cu for <italic>cbb</italic><sub>3</sub>-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during <italic>cbb</italic><sub>3</sub>-Cox assembly.</p>