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.

ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, 1 subunit inhibition has also been described in three bacterial systems, Escherichia coli, Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the 1 subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory ‘extended’ conformation to the ATP-induced non-inhibitory ‘hairpin’ conformation. A recently published crystal structure of the F₁ domain of the C. thermarum TA2.A1 F₁F_o ATP synthase revealed a mutant 1 subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo, and appears to challenge the current dogma on the regulatory role of the 1 subunit. This has prompted a re-examination of present knowledge of the 1 subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving 1 subunit-mediated ATP hydrolysis regulation in a variety of organisms, concluding that the 1 subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.