Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.
The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base.
Holz
M
M
Biophysics Group, Freie Universität Berlin, Federal Republic of Germany.
Drachev
L A
LA
Mogi
T
T
Otto
H
H
Kaulen
A D
AD
Heyn
M P
MP
Skulachev
V P
VP
Khorana
H G
HG
D001216:Asparagine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D010782:Photolysis
D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine