Molecular Analysis of Copper Binding to the Bacterial MFS-type Copper Importer CcoA

Many enzymes require copper (Cu) as a cofactor in proceeding their role in different biological pathways. Copper’s oxidative and reductive abilities make it a good cofactor to accomplish the electron transfer process in reactions. CcoA was discovered in the Gram-negative, facultative photosynthetic model organism Rhodobacater capsulatus. It was found to belong to the Major Facilitator Superfamily (MFS) group of transporter proteins and since then had become the prototype of the newly defined copper uptake porter family. In this capstone project, an antibody that specifically targets the CcoA protein was screened in order to contribute to future molecular analysis of copper binding of the CcoA in the Rhodobacater capsulatus. Different antibody and sample preparation methods were tested in order to optimize the western blot recognition results. Additionally, in order to further investigate the function of the conserved residues in CcoA-like-Transporter family, another aim of the study was focused on the study of the activity of the ccb3-type cytochrome oxidases in a mutant lacking the CcoA homolog protein of the Agrobacterium tumefaciens (fabrum). The study gives insights to the Cu uptake and delivery pathways of the CcoA-like-Transporter in a different bacterial species, and the role of this oxidase on tumor formation in plants. It also emphasizes the function of the conserved histidine residue in these transporters.