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Abstract

The mechanism of proton release to the bulk during the photocycle of bacteriorhodopsin has been studied by absorbance measurements using pyranine as indicator. The initial absorbance change of the indicator is characterized by a sigmoidal shape, reflecting coupling of proton pumping with transfer of protons to the indicator. The time constants of pumping (tau(1) = 20 to 50 mus) and of transfer to the indicator (tau(2) = 0.4 to 1.5 ms) decrease with increasing pyranine concentration, consistent with coupling of an intra- and a bimolecular reaction step. Proton transfer by buffers is dependent on their pK value. The most efficient buffer in the test proved to be borate; in the presence of similar to1 m-M borate protons appeared in the bulk with a time constant of tau(2) approximate to 60 mus. The data are described by simple reaction mechanisms. The rate constants of proton transfer are consistent with a standard dependence on the pK difference between donor and acceptor, but apparently other factors like size and charge of the acceptor contribute as well. The rate constants of proton transfer from bacteriorhodopsin to acceptors with a favorable pK are at the limit of diffusion control. However, the data indicate the existence of a separate intramolecular reaction step for most acceptors, which seems to reflect proton transfer from the release cavity of bacteriorhodopsin to the periphery for acceptors, which cannot approach this cavity because of steric, electrostatic, and/or other factors. Crystal structures of bacteriorhodopsin support the interpretation that the release site is not directly accessible for acceptors such as pyranine. Proton transfer to borate is without barrier and, thus, there is no general diffusion barrier in the proton release of bacteriorhodopsin.