Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF(1) Subunits of the F(1)F(O)-ATPase as Related to the Endosymbiotic Origin of Mitochondria

The F(1)F(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 F(1)F(O)-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF(1)), and chloroplasts (ε and γ disulfide) emerged to block the F(1)F(O)-ATPase activity selectively. In this study, we analyze how these F(1)F(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 F(1)F(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 IF(1) 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.