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

Abstract

The 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 cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions.P. falciparumcyt 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 c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out 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 divergentPlasmodiumcytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

Biogenesis of cytochromescandc1in the electron transport chain of malaria parasites

“…S4), provided further support for this conclusion. Based on our prior observation that P. falciparum HCCS hemylates cyt c in heterologous expression studies in E. coli (16), we propose that HCCS knockdown in parasites impairs holo-cyt c biogenesis and reduces proton pumping by ETC Complex III and/or IV that sensitizes parasites to lethal mitochondrial depolarization by proguanil (Fig. 1).…”

“…The more modest growth phenotype upon knockdown of HCCS compared to HCC1S, despite similar reductions in transcript abundance, is somewhat puzzling and may have multiple underpinnings. Cyt c function, which depends on heme attachment, is essential for ETC function and parasite viability (16). The mild phenotype for HCCS knockdown thus contrasts with the lethal phenotype for cyt c knockdown, the strong expectation of HCCS essentiality based on prior knockout studies (28)(29)(30), and the selective ability of HCCS (and not HCC1S) to hemylate cyt c in heterologous studies in E. coli (16).…”

“…Prior genomewide and targeted knockout studies in P. falciparum and P. berghei reported that both HCCS and HCC1S genes were refractory to disruption and thus likely essential (28)(29)(30), but neither protein has been directly studied in Plasmodium. We first focused on HCC1S, since our prior heterologous expression studies in E. coli suggested that this protein does not hemylate cyt c but were unable to specify a function for this protein in cyt c1 biogenesis (16). To directly test HCC1S function in P. falciparum, we used CRISPR/Cas9 to edit the HCC1S gene in Dd2 parasites to encode a C-terminal HA-FLAG epitope tag fusion and the 10x aptamer/TetR-DOZI system for conditional protein expression (36).…”

“…Prior works have shown that blood-stage parasites can be rescued from defects in the mitochondrial electron transport chain by exogenous addition of the soluble ubiquinone analog, decyl-ubiquinone (dQ) (14)(15)(16). HCC1S knockdown is predicted to impair biogenesis of cyt c1, which is an integral and essential component of ETC Complex III.…”

Plasmodium falciparummitochondrial complex III, the target of atovaquone, is essential for progression to the transmissible sexual stages

“…The importance of PfCIII for Plasmodium survival is well established both based on numerous inhibitor sensitivity and resistance mutant analyses [21][22][23][24][25], and, more recently, through the study of the subunit cytC1 [16]. However, the impact of PfCIII disruption on parasite biology has not been fully characterise.…”