Direct tests of cytochrome <i>c</i> and <i>c</i> <sub>1</sub> functions in the electron transport chain of malaria parasites
Tanya J. Espino-Sanchez, Henry Wienkers, Rebecca G. Marvin, Shai-anne Nalder, Aldo E. García-Guerrero, Peter E. VanNatta, Yasaman Jami‐Alahmadi, Amanda Mixon Blackwell, Frank G. Whitby, James A. Wohlschlegel, Matthew T. Kieber‐Emmons, Christopher P. Hill, Paul A. Sigala
Abstract
The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs ( c and c -2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c -2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c 1 for inducible knockdown. Translational repression of cyt c and cyt c 1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c -2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c -2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c -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 Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.