The 3 × 120° rotary mechanism ofParacoccus denitrificansF1-ATPase is different from that of the bacterial and mitochondrial F1-ATPases

Abstract

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.