Lateral Proton Transfer between the Membrane and a Membrane Protein

Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (similar to 7 x 10(10) s(-1) M-1, compared to the expected value of (1-2) x 10(10) s(-1) M-1), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to similar to 3 x 10(13) s(-1) M-1, which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that. the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.