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.

No abstract

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.

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.

No abstract

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF subunits, prevent the wasteful CCW FF-ATPase activity by blocking γ rotation at the α/β/γ interface of the F portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF, it also binds to the α/β/γ interface of the F of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α/β/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW FF-ATPase rotation without affecting the CW-FF-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdFF-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF) of this unique ATP synthase nanomotor, essential for life.

The F1FO-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 F1FO-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 F1FO-ATPase activity selectively. In this study, we analyze how these F1FO-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 F1FO-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 IF1 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.

No abstract