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Uncovering the transmembrane metal binding site of the novel bacterial major facilitator superfamily-type copper importer CcoA

Journal Article · · mBio (Online)
 [1];  [2];  [3];  [2]
  1. Univ. of Pennsylvania, Philadelphia, PA (United States); University of Pennsylvania
  2. Univ. of Pennsylvania, Philadelphia, PA (United States)
  3. Albert-Ludwigs Univ. of Freiburg, Freiburg (Germany)
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In this study, uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochromecoxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive64Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter.

Research Organization:
Trustees of the Univ. of Pennsylvania, Philadelphia, PA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Grant/Contract Number:
FG02-91ER20052
OSTI ID:
1258013
Journal Information:
mBio (Online), Journal Name: mBio (Online) Journal Issue: 1 Vol. 7; ISSN 2150-7511
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (2)

Cu Transport by the Extended Family of CcoA-like Transporters (CalT) in Proteobacteria journal February 2019
The cbb 3 -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase journal September 2019

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59 BASIC BIOLOGICAL SCIENCES