Mitochondrial proteome of Acanthamoeba delineates aerobic and anaerobic pathways dynamically regulated by oxygen

Acanthamoeba castellanii causes infectious blindness and resides in a key evolutionary outgroup to humans and fungi. Its divergent mitochondria are of outstanding interest due to their predicted aerobic and anaerobic functions with potential for drug targeting. However, a detailed delineation of its bioenergetic machinery, its activities, and their regulation remains lacking. Here, we integrate mitochondrial immunoprecipitation, density gradient purification, mass spectrometry, protein correlation profiling, and microscopy to generate a high-confidence inventory of the Acanthamoeba mitoproteome. The resulting AcMitoCarta contains 1,122 proteins, including 381 lacking readily identifiable homologs in human and yeast mitochondria. Complexome analysis highlights 20 macromolecular assemblies. Complementary proteomic and transcriptomic profiling reveals extensive rewiring of the organelle's bioenergetic machinery by oxygen, including induction of an anaerobic pyruvate:ferredoxin oxidoreductase-to-hydrogenase pathway under anoxia. We experimentally demonstrate that Acanthamoeba can produce H₂ gas under anoxic conditions via a mitochondria-localized, oxygen-labile hydrogenase. AcMitoCarta establishes a framework for dissecting the interplay between aerobic and anaerobic energy metabolism.