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.

The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c 10 ring and the other stator subunits at the F 0 -F 0 dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å . The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ϳ45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356 -12358). In the ATP synthase dimer, the two peripheral stalks are located near the F 1 -F 1 interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.The ATP synthase (F 1 F 0 -ATP synthase; F-ATPase) found in energy-transducing membranes from bacteria, chloroplasts, and mitochondria functions to harness the energy of a transmembrane proton-motive force for the synthesis of ATP from ADP and inorganic phosphate (1). It is now well documented that the energy-coupling mechanism involves a rotary motion of the central stalk of the F 1 F 0 complex driven by proton conduction through F 0 in the forward ATP synthesis direction and an opposite proton-pumping rotation backwards driven by the free energy change of ATP hydrolysis (2-6). The bacterial enzyme possesses a core rotor-stator structure containing eight essential subunits, five in F 1 (␣, ␤, ␥, ␦, and ⑀) and three in F 0 (a, b, and c). This core structure is conserved in eukaryotic mitochondria, but it is complemented with numerous regulatory or supernumerary subunits, most of which are associated with the F 0 proton channel, with the exception of the mitochondrial F 1 -ATPase inhibitor protein (IF 1 ) (7) and a novel inhibitory subunit in the ATP synthase of Paracoccus denitrificans and related ␣-proteobacteria (8). The role of some of the additional F 0 subunits has been unveiled by native gel electrophoresis of the homodimeric mitochondrial ATP synthase (9), by proteomic identification of dimer-specific subunits (10), and by genetic deletion experiments in Saccharomyces cerevisiae, where removal of subunits e and g leads to a loss of the dimeric and oligomeric structures of the complex (11). Remarkably, these deletions (11) or genetic fusions and cross-linking (12) also lead to a loss of the cristae organization of the inner mitochondrial membrane. These observations thus indicate that the dimeric and higher oligomeric species of the ATP synthase exist constitutively in mitochondria promoting formation of mitochondrial cristae (11)(12)(13)(14)(15)(16...

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The subunit is a novel inhibitor of the F 1 F O -ATPase of Paracoccus denitrificans and related ␣-proteobacteria. It is different from the bacterial (⑀) and mitochondrial (IF 1 ) inhibitors. The N terminus of blocks rotation of the ␥ subunit of the F 1 -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 F 1 -ATPase and F 1 -models of P. denitrificans were supported by crosslinking, limited proteolysis, mass spectrometry, and functional data. The final models show that enters into F 1 -ATPase at the open catalytic ␣ E /␤ E interface, and two partial ␥ rotations lock the N terminus of in an "inhibition-general core region," blocking further ␥ rotation, while the globular domain anchors it to the closed ␣ DP /␤ DP interface. Heterologous inhibition of the F 1 -ATPase of P. denitrificans by the mitochondrial IF 1 supported both the modeled binding site at the ␣ DP /␤ DP /␥ interface and the endosymbiotic ␣-proteobacterial origin of mitochondria. In summary, the subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF 1 or the bacterial ⑀ binds. The proposed pawl mechanism is coupled to the rotation of the central ␥ 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 ␣-proteobacteria.

The rotation ofParacoccus denitrificansF1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or belowKm, PdF1showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1molecules suggested that PdF1executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF1is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase fromThermus thermophilus. This contrasts with all other known F1-ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF1. Comprehensive analysis of rotary catalysis of F1from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1and Fodomains of F-ATP synthase. F1motors with more distinctive steps are coupled with proton-conducting Forings with fewer proteolipid subunits, giving insight into the design principle the F1Foof ATP synthase.

The biological roles of the three natural FF-ATPase inhibitors, ε, ζ, and IF, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF, but in the FF-ATPase of Paracoccus denitrificans (PdFF), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdFF-ATPase activity without affecting the PdFF-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdFF-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.