A MICROSECOND KINETIC STUDY OF THE PHOTOGENERATED MEMBRANE POTENTIAL OF BACTERIORHODOPSIN WITH A FAST RESPONDING DYE

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Abstract
Abstract— Bacteriorhodopsin is a light activated proton pump which generates proton and electric gradients across the cytoplasmic membrane of Halobacterium halobium. In this study, a dye whose fluorescence intensity responds rapidly to membrane potential was used to follow the evolution of the potential on liposomes reconstituted with bacteriorhodopsin, in the microseconds time domain. By comparing the formation kinetics of the potential to those of the long‐lived intermediate species in the bacteriorhodopsin photocycle, M412, both in H2O and 2H2O suspensions, we can draw the following conclusion: the electric potential onset time is 20 μs after initiation of the illumination. The triggering of the potential is not the formation of the M412 intermediate, which was hitherto considered to be the first species in the bacteriorhodopsin cycle which has an unprotonated Schiff base linkage at the retinal chromophore. Rather, the potential forms at the transition of the L550 intermediate to the species X which precedes M412 or even at the preceding conversion of K590 to L550.