Changes in the structure of mitochondrial processing peptidase driven by adaptation to anaerobiosis

Transition of eukaryotes from oxygen-rich to oligoxic or anoxic environments drove profound physiological changes, particularly in mitochondria. Low oxygen led to the loss of respiratory complexes that generate the inner mitochondrial membrane (IM) electrochemical gradient, as seen in anaerobic types of mitochondria, like the hydrogenosomes in Trichomonas vaginalis. Besides energy metabolism, IM potential is critical for organelle biogenesis, particularly the import of matrix preproteins. These preproteins contain positively charged N-terminal targeting sequences (NTSs) that facilitate import via the TIM23 translocase. Upon translocation, NTS is cleaved by mitochondrial processing peptidase (MPP), a zinc metallopeptidase composed of α and β subunits. The α-MPP glycine-rich loop (GRL) recognizes and delivers the presequence to the β-MPP active site. Hydrogenosomal NTSs are shorter and less positively charged, reflecting reduced IM potential. Using X-ray crystallography, modeling, and mutagenesis, we reveal distinctive structural adaptations of the hydrogenosomal processing peptidase (HPP). Compared to MPP, HPP has a central chamber about half the size, with predominantly electropositive and neutral surface charges. The α-HPP GRL features a reduced conserved glycine motif and is positioned closer to the β-HPP active site. Enzymatic assays of β-HPP mutants revealed a novel substrate interaction site involved in Zn²⁺ dependent catalysis. These structural adaptations match the unique properties of hydrogenosomal NTSs, optimizing their processing under anaerobic conditions. They illustrate how adaptation to anaerobiosis drives protein structural evolution to sustain life in oxygen-poor environments.