The ζ subunit of the F1FO-ATP synthase of α-proteobacteria controls rotation of the nanomotor with a different structure.

The ζ subunit is a novel natural inhibitor of the α-proteobacterial F1FO-ATPase described originally in Paracoccus denitrificans. To characterize the mechanism by which this subunit inhibits the F1FO nanomotor, the ζ subunit of Paracoccus denitrificans (Pd-ζ) was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite-activated PdF1-ATPase with an apparent IC50 of 270 nM by a mechanism independent of the ε subunit. The inhibitory region of the ζ subunit resides in the first 14 N-terminal residues of the protein, which protrude from the 4-α-helix bundle structure of the isolated ζ subunit, as resolved by NMR. Cross-linking experiments show that the ζ subunit interacts with rotor (γ) and stator (α, β) subunits of the F1-ATPase, indicating that the ζ subunit hinders rotation of the central stalk. In addition, a putatively regulatory nucleotide-binding site was found in the ζ subunit by isothermal titration calorimetry. Together, the data show that the ζ subunit controls the rotation of F1FO-ATPase by a mechanism reminiscent of, but different from, those described for mitochondrial IF1 and bacterial ε subunits where the 4-α-helix bundle of ζ seems to work as an anchoring domain that orients the N-terminal inhibitory domain to hinder rotation of the central stalk.