J. Biol. Chem., Vol. 267, Issue 36, 25730-25733, 12, 1992
Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the Schiff base
S Subramaniam, DA Greenhalgh and HG Khorana
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
In bacteriorhodopsin Asp85 has been proposed to function both as a negative
counterion to the Schiff base and as proton acceptor in the early stages of
the photocycle. To test this proposal further, we have replaced Asp85 by
His. The rationale for this replacement is that although His can function
as a proton acceptor, it cannot provide a negative charge at residue 85 to
serve as a counterion to the protonated Schiff base. We show here that the
absorption spectrum of the D85H mutant is highly sensitive to the pH of the
external medium. From spectroscopic titrations, we have determined the
apparent pK for deprotonation of the Schiff base to be 8.8 +/- 0.1 and the
apparent pK for protonation of the His85 side chain to be approximately
3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the
His side chain is deprotonated, the D85H mutant is completely inactive in
proton transport. Time-resolved studies show that there is no detectable
formation of an M-like intermediate in the photocycle of the D85H mutant.
These experiments show that the presence of a neutral proton-accepting
moiety at residue 85 is not sufficient for carrying out light-driven proton
transport. The requirements at residue 85 are therefore for a group that
serves both as a negatively charged counterion and as a proton acceptor.
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