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 ( co - ike ransporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for -type cytochrome oxidase ( -Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met xxxMet and His xxxMet motifs in its TMH7 and TMH8, respectively. Of these residues, Met and His are essential for Cu uptake and -Cox production, whereas Met and Met 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 and Met completely abolishes Cu uptake and -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 located at the end of TMH2, the Cys on a surface loop between TMH7 and THM8, and the C 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 , possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for -Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.