TIME-RESOLVED SURFACE-CHARGE CHANGE ON THE CYTOPLASMIC SIDE OF BACTERIORHODOPSIN

The pH-sensitive dye 5-iodoacetamidofluorescein was covalently bound t o a single cysteine residue introduced by site-directed mutagenesis in position 101 on the cytoplasmic surface or in position 130 on the ext racellular surface of the proton pump bacteriorhodopsin. Using time-re solved absorption spectroscopy at 495 nm a transient increase was obse rved in the apparent pK of the dye attached at residue 101. At pH 7.3 the rise and decay times of this pK-change (similar to 2 ms and simila r to 60 ms) correlate well with decay times observed for the M and O i ntermediates and with the proton uptake time. Interpreting the pg-incr ease of +0.18 pH-unit in terms of a transiently more negative surface charge density, we calculate a change of -0.80 elementary charge per b acteriorhodopsin at the cytoplasmic surface. It is likely that this ch arge change is due to the transient deprotonation of aspartate-96. Wit h the label in position 130 on the extracellular surface no transient pK-shift was detected.