Expression and copper binding studies of a Plasmodium falciparum protein with Cox19 copper binding motifs

Copper can exist in an oxidized and a reduced form, which enables the metal to play essential roles as a catalytic co-factor in redox reactions in many organisms. Copper confers redox activity to the terminal electron transport chain cytochrome c oxidase protein. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11 in association with Cox19. When copper is chelated in growth medium, Plasmodium fal-ciparum parasite development in infected red blood cells is inhibited and excess copper is toxic for the parasite. The gene of a 26 kDa Plasmodium falciparum PfCox19 protein with two Cx9C Cox19 copper binding motifs, was cloned and expressed as a 66 kDa fusion protein with maltose binding protein and affinity purified (rMBP- PfCox19). rMBP-PfCox19 bound copper measured by: a bicinchoninic acid release assay; an in vivo bacterial host growth inhibition assay; ascorbate oxidation inhibition and differential scanning fluorimetry. The native protein was detected by antibodies raised against rMBP-PfCox19. PfCox19 binds copper and is predicted to associate with PfCox11 in the insertion of copper into the CuB site of Plasmodium cytochrome c oxidase. Characterisation of the proteins involved in Plasmodium spp. copper metabolism will help us understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.