Rapid light-induced surface charge changes in bacteriorhodopsin

Abstract

Purple membranes from Halobacterium halobium contain bacteriorhodopsin which is a light-driven electrogenic proton pump [ 11. After light excitation the chromophore which is retinal-bound through a Schiff base to a lysyl residue in the opsin, forms several photointermediates (designated K, L, M, 0) returning to the ground state at the end of the cycle. The major photochemical and biochemical events involve isomerization of the retinal from all-pans to 13-cis [2] and dissociation of a proton from the retinal Schiff base [3]. The electrogenic transfer of protons should also involve charge separation within and across the protein. Measurements of photoelectric transient response in oriented purple membranes were assumed to reflect these changes. The experimental systems included either purple membranes or bacteriorhodopsin liposomes attached to lipid-impregnated filters, lipid bilayer, teflon films or multilayers of purple membranes [4-71. We have used molecular probes to monitor surface potential changes in purple membranes [8,9]. Spin probes were used to measure the photostationary and pulse-induced surface potential charges. However, because of the relatively low sensitivity and the slow response of EPR, the measurements were performed only under experimental conditions which induced a slowdown in the photocycle. Here, we have developed a highly sensitive optical method having a fast time resolution. The method involves measurements of absorption changes in a pH indicator adsorbed to the purple membranes suspended in a highly buffered solution. It was shown [lo] that fixed charges alter the apparent pK, of adsorbed indicators. Measurements of the kinetics of