Abstract
The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599-608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F1-ATPase and F1-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1-ATPase at the open catalytic αE/βE interface, and two partial γ rotations lock the N terminus of ζ in an "inhibition-general core region," blocking further γ rotation, while the ζ globular domain anchors it to the closed αDP/βDP interface. Heterologous inhibition of the F1-ATPase of P. denitrificans by the mitochondrial IF1 supported both the modeled ζ binding site at the αDP/βDP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF1 or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.