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.
Highlights
Copper is an essential, yet potentially toxic micronutrient, which is required for the catalytic activity of many important enzymes, like cytochrome oxidases (Cox) or Cu-Zn superoxide dismutases
The CPC motif (TM-MBS1) is located in the transmembrane helix (TM) 4 and the NMS motif (TM-MBS2) is formed by an asparagine residue from TM5 together with methionine and serine residues from TM8. Both transmembrane helices (TM)-metal binding sites (MBSs) are conserved among Cu-transporting P1B-ATPases (Arguello et al, 2007; Smith et al, 2014) and are necessary to drive the outward transport of copper (Mandal et al, 2004)
Previous studies had demonstrated that CcoI is essential for the assembly and activity of cbb3-Cox in bacteria, including R. capsulatus (Koch et al, 2000; Kulajta et al, 2006; Utz et al, 2019), Bradyrhizobium japonicum (Preisig et al, 1996) and Rubrivivax gelatinosus (Hassani et al, 2010)
Summary
Copper is an essential, yet potentially toxic micronutrient, which is required for the catalytic activity of many important enzymes, like cytochrome oxidases (Cox) or Cu-Zn superoxide dismutases. Cu homeostasis is tightly controlled by the coordinated action of Cu transporters and Cu chaperones to maintain a sufficient Cu quota for cuproprotein biogenesis while preventing accumulation of excess Cu in the cytosol (Rosenzweig and O'Halloran, 2000; Rensing and Grass, 2003; Banci et al, 2010a; Arguello et al, 2016) Central to this coordination are P1B-type ATPases, a ubiquitous class of transition metal exporters, located in the cytoplasmic membrane of bacteria, the basolateral membrane of enterocytes or the trans-Golgi network of hepatocytes and brain cells (Crisponi et al, 2010; Smith et al, 2014; Andrei et al, 2020). Due to the potential toxicity of Cu and the virtual absence of free Cu in the bacterial cytosol, the catalytic activity of P1B-type ATPases is thought to be further influenced by Cu transfer reactions from cytosolic Cu chaperones to their MBSs (Arguello et al, 2016)
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