Mechanically driven proton conduction in single δ-free F0F1-ATPase

In order to observe mechanically driven proton flux in F0F1-ATPase coupled with artificial driven rotation on F1 simultaneously, a double channel observation system was established. An artificial δ-free F0F1-ATPase was constructed with α3, β3, ε, γ, and cn subunits as rotator and a, b2 as stator. The chromatophore was immobilized on the glass surface through biotin–streptavidin–biotin system, and the magnetic bead was attached to the β subunit of δ-free F0F1-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F0 and F1 is about 4.15 ± 0.2H+/rev when the magnetic field rotated at 0.33 Hz (rps).