A new family of TonB-dependent copper transporters linked to respiratory oxidase function

Copper is an essential metal notably found in respiration complexes for its redox properties. It is also toxic hence its cellular trafficking is tightly controlled. Bacteria have developed a number of defense systems against copper excess, but its acquisition pathways remain poorly characterized. Ubiquitous in Gram-negative bacteria, TonB-dependent transporters (TBDTs) are outer membrane β-barrel proteins that mediate the proton motive force-dependent import of various nutrients to the periplasm. Here, we characterized a TBDT that imports copper in the whooping cough agent Bordetella pertussis, CrtA^Bp (formerly BfrG), which is a prototype of a new subfamily of TBDTs. Our data indicate that CrtA^Bp is dedicated to the import of copper for heme-copper respiratory oxidoreductases. We revealed that CrtA^Bp imports chelated rather than free copper, solved the crystal structure of CrtA^Bp and identified a conserved ligand binding site. By combining bacterial growth experiments, biophysical approaches and AlphaFold3 modeling we sketched out the features of copper-ligand complexes for CrtA^Bp. In contrast with ferrisiderophore-specific TBDTs, no high-affinity chalkophore ligand of CrtA^Bp could be identified, implying two nonmutually exclusive models. In the host, CrtA^Bp might use a xenometallophore produced by another species present in the same niche to acquire copper. In vitro however, CrtA appears not to have high-affinity ligands but to import copper chelated by small molecules notably harboring carboxylate groups, which might represent a paradigm of 'scavenger' TBDTs with low ligand selectivity. We identified an essential, invariant histidine residue that might serve as a selectivity filter for copper-chelate complexes.