Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites

Abstract

ABSTRACTThe mitochondrial electron transport chain (ETC) ofPlasmodiummalaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cytchomologs (candc-2) with unusually sparse sequence identity and uncertain fitness contributions.P. falciparumcytc-2 is the most divergent eukaryotic cytchomolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cytchomologs and the related cytc1for inducible knockdown. Translational repression of cytcand cytc1was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cytc-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cytcfor ETC function. Biochemical and structural studies revealed that both cytcandc-2 are hemylated by holocytochromecsynthase, but UV-vis absorbance and EPR spectra strongly suggest that cytc-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergentPlasmodiumcytochromecwith molecular adaptations that defy a conserved role in eukaryotic evolution.SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion ofPlasmodiummalaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochromecproteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cytcis essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.