RELATIONSHIP OF PROTON RELEASE AT THE EXTRACELLULAR SURFACE TO DEPROTONATION OF THE SCHIFF-BASE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The surface potential of purple membranes and the release of protons d uring the bacteriorhodopsin photocycle have been studied with the cova lently linked pH indicator dye, fluorescein. The titration of acidic l ipids appears to cause the surface potential to be pH-dependent and ca uses other deviations from ideal behavior, if these anomalies are negl ected, the appearance of protons can be followed by measuring the abso rption change of fluorescein bound to various residues at the extracel lular surface. Contrary to widely held assumption, the activation enth alpies of kinetic components, deuterium isotope effects in the time co nstants, and the consequences of the D85E, F208R, and D212N mutations demonstrate a lack of direct correlation between proton transfer from the buried retinal Schiff base to D85 and proton release at the surfac e. Depending on conditions and residue replacements, the proton releas e can occur at any time between the protonation of D85 and the recover y of the initial state. We conclude that once D85 is protonated the pr oton release at the extracellular protein surface is essentially indep endent of the chromophore reactions that follow. This finding is consi stent with the recently suggested version of the alternating access me chanism of bacteriorhodopsin, in which the change of the accessibility of the Schiff base is to and away from D85 rather than to and away fr om the extracellular membrane surface.