Redefining the role of the Plasmodium heme detoxification protein: From hemozoin formation to mitochondrial protein synthesis

Throughout their intraerythrocytic development, malaria parasites digest up to 80% of the host cell’s hemoglobin within a specialized degradative compartment known as the digestive vacuole. This process releases heme, which is detoxified by sequestration into bioinert hemozoin crystals. Although heme biomineralization is essential for blood-stage survival and a validated drug target, its underlying mechanisms remain unclear. Initially identified as a potent inducer of β-hematin crystallization in vitro , the parasite’s H eme D etoxification P rotein (HDP) has been proposed to execute a similar role in the formation of hemozoin crystals in cellulo . Here, we investigate the function of HDP in live Plasmodium falciparum parasites, integrating experimental genetic approaches with quantitative microscopy, cellular bioenergetics and whole-proteome profiling. Endogenous tagging revealed that HDP localizes to the mitochondrion rather than the digestive vacuole. Conditional inactivation of HDP resulted in a gradual loss of mitochondrial membrane potential, preceding developmental arrest. Bypassing the essential role of the respiratory chain in pyrimidine biosynthesis – either through exogenous electron acceptors or expression of a ubiquinone-independent dihydroorotate dehydrogenase – rescued HDP-deficient parasites, indicating a role in maintaining respiratory chain activity. Consistent with this, electron flow through complex IV was abolished in rescued HDP-null parasites, increasing sensitivity to proguanil, an antimalarial that synergizes with respiratory chain inhibitors. We found that loss of HDP leads to a marked reduction of complexes III and IV, whose integrity depends on mitochondrial protein biosynthesis. Integration of quantitative proteomic data with structure-guided homology modelling supports a role for HDP as part of the large mitoribosomal subunit at the inter-subunit contact site. By contrast, neither the quantity nor morphology of hemozoin was affected by the loss of HDP, and sensitivity to the hemozoin-targeting drug chloroquine remained unchanged. Together, these findings challenge previous models linking HDP to hemozoin formation and instead reveal an essential role for HDP in mitochondrial protein biosynthesis.