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

Mariel Zarco-Zavala, Ryo Watanabe, Duncan G.G. McMillan, Toshiharu Suzuki, Hiroshi Ueno, Francisco Mendoza-Hoffmann, José J. García-Trejo, Hiroyuki Noji

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Abstract

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 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.

Original languageEnglish
Pages (from-to)29647-29657
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number47
DOIs
Publication statusPublished - 2020

Keywords

  • F1-ATPase
  • Rotation
  • Single-molecule analysis
  • ζ-subunit

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