The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb 3-Type Cytochrome Oxidase Biogenesis

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

doi:10.3389/fmicb.2021.712465

The CopA2-Type P_1B-Type ATPase CcoI Serves as Central Hub for cbb ₃-Type Cytochrome Oxidase Biogenesis

Front Microbiol. 2021 Sep 13:12:712465. doi: 10.3389/fmicb.2021.712465. eCollection 2021.

Authors

Affiliations

¹ Institut für Biochemie und Molekularbiologie, ZBMZ, Faculty of Medicine, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
² Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
³ Institute for Biochemistry, Faculty of Chemistry and Pharmacy, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
⁴ Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
⁵ Faculty of Chemistry and Pharmacy, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
⁶ Department of Biology, University of Pennsylvania, Philadelphia, PA, United States.

Abstract

Copper (Cu)-transporting P_1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P_1B-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 cbb ₃-type cytochrome oxidase (cbb ₃-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 cbb ₃-Cox. CcoI and cbb ₃-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 cbb ₃-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb ₃-Cox assembly.

Keywords: CcoI; CopZ; P1B-type ATPases; Rhodobacter capsulatus; SenC; cbb3-type cytochrome c oxidase biogenesis; solid-supported membrane electrophysiology.