Inhibitory to non-inhibitory evolution of the subunit of the FF-ATPase of and -proteobacteria as related to mitochondrial endosymbiosis.

The subunit is a potent inhibitor of the FF-ATPase of (PdFF-ATPase) and related -proteobacteria different from the other two canonical inhibitors of bacterial () and mitochondrial (IF) FF-ATPases. mimics mitochondrial IF in its inhibitory N-terminus, blocking the PdFF-ATPase activity as a unidirectional pawl-ratchet and allowing the PdFF-ATP synthase turnover. is essential for the respiratory growth of , as we showed by a knockout. Given the vital role of ζ in the physiology of , here, we assessed the evolution of across the -proteobacteria class. Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several subunits, we confirmed the conservation of the inhibitory N-terminus of and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant subunits from several -proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. The results show that evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the inhibitory function was diminished or lost in some symbiotic α-proteobacteria where is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the subunit of (Sm-) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm- cannot exert homologous inhibition. However, it is still able to inhibit the PdFF-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial , the data confirm that the primary inhibitory function of the α-proteobacterial FF-ATPase was transferred from to and that ζ, ε, and IF evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from -proteobacteria, are also discussed.