LAUSR

Significance F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), spreading across all kingdoms of life. Despite remarkable conservation of its basic structure and function, biophysical studies have revealed discrete differences in the rotary mechanisms of bacterial and eukaryotic F1-ATPases (the catalytic portions of the enzymes). Here, we analyzed the rotational dynamics of Paracoccus denitrificans F1 (PdF1), a bacterial F1-ATPase that exhibits high homology with the core functional subunits of its mitochondrial counterpart. Notably, PdF1 possesses a simplified chemomechanical scheme different from that of all other F1-ATPases. Our results reveal an unexpected diversity in the chemomechanical coupling cycle of the F1-ATPase machinery and show that features such as homology or phylogenetic relationship cannot uniquely define the rotary scheme pattern. The rotation of Paracoccus denitrificans F1-ATPase (PdF1) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below Km, PdF1 showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF1 executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF1 molecules suggested that PdF1 executes 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 PdF1 is principally a three-stepping motor similar to bacterial vacuolar (V1)-ATPase from Thermus 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 F1 from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F1 and Fo domains of F-ATP synthase. F1 motors with more distinctive steps are coupled with proton-conducting Fo rings with fewer proteolipid subunits, giving insight into the design principle the F1Fo of ATP synthase.