We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.

Department of Biochemistry and Biophysics The Arrhenius Laboratories for Natural Sciences Stockholm University SE 106 91 Stockholm Sweden

Department of Biophysical Chemistry J Heyrovský Institute of Physical Chemistry of the A S C R v v i Prague Czech Republic

Department of Chemistry and Biochemistry University of Bern 3012 Bern Switzerland

Experimental Biomolecular Physics Department of Applied Physics Royal Institute of Technology SE 106 91 Stockholm Sweden

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