The F₁F_O-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F₁F_O-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF₁), and chloroplasts (ε and γ disulfide) emerged to block the F₁F_O-ATPase activity selectively. In this study, we analyze how these F₁F_O-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F₁F_O-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF₁ likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria.

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.