Inhibitory to non-inhibitory evolution of the ? subunit of the F₁F_O-ATPase of Paracoccus denitrificans and a-proteobacteria as related to mitochondrial endosymbiosis

Introduction: The ? subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related a-proteobacteria different from the other two canonical inhibitors of bacterial (e) and mitochondrial (IF1) F1FO-ATPases. ? mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ? is essential for the respiratory growth of P. denitrificans, as we showed by a ?? knockout. Given the vital role of ? in the physiology of P. denitrificans, here, we assessed the evolution of ? across the a-proteobacteria class.Methods: 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 a-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic a-proteobacteria.Results and discussion: The results show that ? evolved, preserving its inhibitory function in free-living a-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ? inhibitory function was diminished or lost in some symbiotic a-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 Sinorhizobium meliloti (Sm-?) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended a-helical inhibitory N-terminus conformation, thus explaining why the Sm-? cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of a-proteobacterial e, the data confirm that the primary inhibitory function of the a-proteobacterial F1FO-ATPase was transferred from e to ? and that ?, e, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from a-proteobacteria, are also discussed.