Evidence for an early evolutionary emergence of γ-type carbonic anhydrases as components of mitochondrial respiratory complex I

Background: The complexity of mitochondrial complex I (CI; NADH: ubiquinone oxidoreductase) has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to gamma-type carbonic anhydrases (gamma CA) have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution. Results: We have performed a tandem mass spectrometric characterization of CI from the amoeboid protozoon Acanthamoeba castellanii. Among the proteins identified were two gamma CA homologs, AcCa1 and AcCa2, demonstrating that gamma CA proteins are not specific to plants/green algae. In fact, through bioinformatics searches we detected gamma CA homologs in diverse protist lineages, and several of these homologs are predicted to possess N-terminal mitochondrial targeting peptides. Conclusions: The detection of gamma CAs in CI of Acanthamoeba, considered to be a closer relative of animals and fungi than plants, suggests that gamma CA proteins may have been an ancestral feature of mitochondrial CI, rather than a novel, plant-specific addition. This assertion is supported by the presence of genes encoding gamma CAs in the nuclear genomes of a wide variety of eukaryotes. Together, these findings emphasize the importance of a phylogenetically broad characterization of CI for elucidating CI evolution in eukaryotes.