Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.
Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.
Heberle
J
J
Hahn-Meitner-Institut, BENSC-N1, Berlin, Germany.
Oesterhelt
D
D
Dencher
N A
NA
D001216:Asparagine / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics*; D005973:Glutamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011522:Protons
D011522:Protons; D005973:Glutamine; D001436:Bacteriorhodopsins; D001216:Asparagine