Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

Abstract

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (CuB) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the CuB atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the CuB site of cbb3-type cytochrome c oxidase (cbb3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb3- and aa3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb3-Cox and not that of aa3-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb3-Cox biogenesis, establishing that distinct copper uptake systems provide the CuB atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions.

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

“…On the other hand, a deletion of copZ in B. subtilis did not influence aa 3 ‐type cytochrome oxidase ( aa 3 ‐Cox) activity (Radford et al , ), in agreement with the occurrence of dedicated Cu chaperones, like CtaG or Sco1, for aa 3 ‐Cox assembly (Bengtsson et al , ; Hill and Andrews, ). However, in Rhodobacter species, the cbb 3 ‐Cox and aa 3 ‐Cox assembly pathways do not engage identical Cu uptake and trafficking conduits (Khalfaoui‐Hassani et al , ; ). The cbb 3 ‐Cox activity in the Δ copZ strain is reduced to about 70% of the wild type activity, whereas the deletion of copA does not reduce cbb 3 ‐Cox activity, and the expression of a plasmid‐encoded copy of copA in the Δ ccoI strain does not rescue its cbb 3 ‐Cox assembly defect.…”

Handling of nutrient copper in the bacterial envelope

“…Indeed, potential roles in Cu insertion for other accessory components such as CcoG, CcoH, and CcoS (at least for the cbb3 oxidase) have been proposed, which may reflect their role in regulating such maturation checkpoints but mechanistic data are currently lacking. 93,110,111 Like the other extracytoplasmic cuproenzymes described in this review, haem-Cu oxidases also appear to utilise nutrient Cu that has been routed via the cytoplasm, first via a major facilitator superfamily (MFS)-type transporter named CcoA that putatively imports Cu into the cytoplasm 112,113 and subsequently via a Cu efflux pump (CcoI or CtpA) 31,111,114,115 ( Figure 6). Deletion of each of these transporters leads to decreases in the activities of CuB and/or CuA-containing cytochrome oxidase activities, but these are, to some extent, alleviated by supplementation with Cu salts.…”

Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Supercomplex and Electronic Communication Between the Complexes

“…R. capsulatus strains were grown chemoheterotrophically under semi-aerobic/dark conditions at 35 °C on enriched (MPYE) medium, supplemented as needed with gentamicin, kanamycin, spectinomycin, or tetracycline at 3, 10, 10 or 2.5 μg/mL, respectively. Colonies were stained for cbb 3 -type CIV activity by incubating plates with a 1:1 (v/v) mixture of 35 mM 1-naphtol and 30 mM N,N-dimethyl-1,4-phenylenediamine (NADIstaining) 61 .…”

“…A similar strain, M7G-CBC1 that lacks both CIII 2 and the CcoP subunit but overproduces the CcoN and CcoO subunits of CIV 31 , was also used. The plasmids pYO76 and pYO92 encoding the bipartite and tripartite SCs, respectively, were conjugated into YO12 and M7G-CBC1 using triparental mating to yield pYO76/YO12 and pYO92/M7G-CBC1 strains as in 61 for protein purification. Protein analyses.…”