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 the 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. Here researchers studied the evolution of these inhibitory proteins finding that they evolved independently by convergent evolution, this means that they emerged for instance as the wings of birds and bats, meaning that they appeared in different times and species, but converged in a similar function although with different structures, and in the case of these inhibitor proteins, somehow converging in the same binding site to inhibit the F-ATPase activity. The finding that some of these inhibitory proteins has lost the inhibitory function such as the ε subunit in α-proteobacetria, and in the case of the ζ subunit it has also lost the inhibitory function and even disappeared in some symbiotic or parasitic α-protoebacteria very closely related to the origin of mitochondria, indicate that researchers can propose ε and ζ genes of the ATP synthase as tracers of the α-proteobacteria that was chosen by evolution as the pre-endosymbiont that eventually became the actual mitochondria. Some implications on the identity of the putative mitochondrial pre-endosymbiont, and the putative steps in mitochondrial evolution in concordance with the endosymbiotic theory as supported by Lynn Margulis and with other recent evolutionary studies, as well as some applications in the design of future antimicrobials targeting the α-proteobacterial ATP synthase are also discussed. This video and abstract are summarizing the review and experimental paper by Mendoza-Hoffman et al. Microorganisms, 2022.