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.