Inhibitory to non-inhibitory evolution of the ζ subunit of the F(1)F(O)-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis

Introduction: The ζ subunit is a potent inhibitor of the F(1)F(O)-ATPase of Paracoccus denitrificans (PdF(1)F(O)-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF(1)) F(1)F(O)-ATPases. ζ mimics mitochondrial IF(1) in its inhibitory N-terminus, blocking the PdF(1)F(O)-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF(1)F(O)-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ 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 ζ subunit of Sinorhizobium meliloti (Sm-ζ) 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-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF(1)F(O)-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F(1)F(O)-ATPase was transferred from ε to ζ and that ζ, ε, and IF(1) evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.