Résumé
The rotary motor F₁ ATPase from the thermophilic Bacillus PS3 (TF₁) is one of the best-studied of all molecular machines. F₁- ATPase is the part of the enzyme F₁F₀-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F₁-ATPase from Escherichia coli (EF₁) is governed by the same mechanism as TF₁ under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF₁ molecules, we characterized the ATP-binding, catalytic and inhibited states of EF₁. We also show that the ATP-binding and catalytic states are separated by 35 ± 3°. At room temperature, chemical processes occur faster in EF₁ than in TF₁, and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.