Biogenesis of Cytochromes <i>c</i> and <i>c</i> ₁ in the Electron Transport Chain of Malaria Parasites

<i>Plasmodium</i> malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and is a key antimalarial drug target. ETC function requires cytochromes <i>c</i> and <i>c</i>₁, which are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome <i>c</i> synthase (HCCS) that binds heme and the apo cytochrome to facilitate the biogenesis of the mature cytochrome <i>c</i> or <i>c</i>₁. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, <i>Plasmodium</i> parasites are like yeast and encode two separate HCCS homologues thought to be specific for heme attachment to cyt <i>c</i> (HCCS) or cyt <i>c</i>₁ (HCC₁S). To test the function and specificity of <i>Plasmodium falciparum</i> HCCS and HCC₁S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC₁S knockdown selectively impaired cyt <i>c</i>₁ biogenesis and caused lethal ETC dysfunction that was not reversed by the overexpression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in <i>Escherichia coli</i> of cyt <i>c</i> hemylation by <i>P. falciparum</i> HCCS and HCC₁S strongly suggest that both homologues are essential for mitochondrial ETC function and have distinct specificities for the biogenesis of cyt <i>c</i> and <i>c</i>₁, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.