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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 47; pp. 29647 - 29657
• Main Authors: Zarco-Zavala, Mariel, Watanabe, Ryo, McMillan, Duncan G. G., Suzuki, Toshiharu, Ueno, Hiroshi, Mendoza-Hoffmann, Francisco, García-Trejo, José J., Noji, Hiroyuki
• Format: Journal Article
• Language: English
• Published: Washington National Academy of Sciences 24.11.2020
• Subjects: Adenosine diphosphate; Adenosine triphosphatase; Adenosine triphosphate; AMP; Angular position; ATP; ATP synthase; Binding; Biological Sciences; Catalysis; Dwell time; F1-ATPase; Hydrolysis; Mitochondria; Paracoccus denitrificans; Rotation; Stepping motors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2003163117

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 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.