Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT ( C co A - l ike T ransporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met 233 xxxMet 237 and His 261 xxxMet 265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met 233 and His 261 are essential for Cu uptake and cbb 3 -Cox production, whereas Met 237 and Met 265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met 237 and Met 265 completely abolishes Cu uptake and cbb 3 -Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys 49 located at the end of TMH2, the Cys 247 on a surface loop between TMH7 and THM8, and the C 367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo , possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb 3 -Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function.