Espino-Sanchez, T. J. | Search bioRxiv

bioRxiv2025

A Divergent Cytochrome c in Malaria Parasites with an Anomalously Low Redox Potential

Eukaryotic cytochrome (cyt) c is a highly conserved mitochondrial protein central to cellular respiration, featuring a covalently attached hexacoordinate heme whose redox potential is tuned by axial His/Met ligands and surrounding residues to support electron transport chain (ETC) function. We have identified an unrecognized lineage of eukaryotic cyt c homologs in Apicomplexa, a phylum of intracellular pathogens that includes Plasmodium falciparum malaria parasites. P. falciparum cyt c-2 (Pfcyt c-2) exemplifies this divergent lineage and has an unusual pentacoordinate heme despite conservation of His/Met ligands. We determined that Pfcyt c-2 has a redox potential of -278 mV that is over 500 mV lower than canonical cyt c homologs (+250 mV) and contradicts a conserved ETC role. This anomalous redox potential is lower than any natural monoheme c-type cyt. Nevertheless, Pfcyt c-2 displays canonical thermostability and low-level peroxidase activity, while showing signs of elevated structural heterogeneity. These results reveal a new clade of eukaryotic cyt c variants with divergent biochemical properties and biological roles, opening new scaffolds for mechanistic discovery and redox engineering.

biochemistry↗

bioRxiv2023

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

The mitochondrial electron transport chain (ETC) of Plasmodium malaria 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. 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 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 divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria 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 cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is 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.

microbiology↗