In order to observe mechanically driven proton flux in F0F1-ATPase coupled with artificial driven rotation on F, simultaneously, a double channel observation system was established. An artificial delta-free F0F1-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-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 F-0 and F-1 is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps). (c) 2006 Elsevier Inc. All rights reserved.