Direct tests of cytochrome <i>c</i> and <i>c</i>₁ functions in the electron transport chain of malaria parasites.

Abstract

The mitochondrial electron transport chain (ETC) of <i>Plasmodium</i> malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt <i>c</i> homologs (<i>c</i> and <i>c</i>-2) with unusually sparse sequence identity and uncertain fitness contributions. <i>P. falciparum</i> cyt <i>c</i>-2 is the most divergent eukaryotic cyt <i>c</i> homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt <i>c</i> homologs and the related cyt <i>c</i>₁ for inducible knockdown. Translational repression of cyt <i>c</i> and cyt <i>c</i>₁ was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt <i>c</i>-2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt <i>c</i> for ETC function. Biochemical and structural studies revealed that both cyt <i>c</i> and <i>c</i>-2 are hemylated by holocytochrome <i>c</i> synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt <i>c</i>-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 divergent <i>Plasmodium</i> cytochrome <i>c</i> with molecular adaptations that defy a conserved role in eukaryotic evolution.