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 K_m, PdF1 showed 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 PdF1 molecules 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 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 F₁from different species, including PdF1, suggests a clear trend in the correlation between the numbers of rotary steps of F₁and Fo domains of F-ATP synthase. F₁motors 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.

The F₁-ATPase is the catalytic portion of the F_oF₁ ATP synthase and acts as a rotary molecular motor when it hydrolyzes ATP. Two decades have passed since the single-molecule rotation assay of F₁-ATPase was established. Although several fundamental issues remain elusive, basic properties of F-type ATPases as motor proteins have been well characterized, and a large part of the reaction scheme has been revealed by the combination of extensive structural, biochemical, biophysical, and theoretical studies. This review is intended to provide a concise summary of the fundamental features of F₁-ATPases, by use of the well-described model F₁ from the thermophilic Bacillus PS3 (TF₁). In the last part of this review, we focus on the robustness of the rotary catalysis of F₁-ATPase to provide a perspective on the re-designing of novel molecular machines.