DECOUPLING OF PHOTO-CYCLE 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 nati ve 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 n ot only accepts the proton released by the Schiff's base but also regu lates very efficiently proton transfer within the proton release chain . Furthermore, Asp85 might be the primary but is not the only proton a cceptor/donor group in the release pathway. The Asp85 --> Glu substitu tion also affects the proton reuptake reaction at the cytoplasmic side , although Asp85 is located in the proton release pathway. Proton upta ke is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechani sm between Asp85 and the proton uptake pathway in bacteriorhodopsin.