Biophysical Journal
1996 January, 70(1); 473-481
Sergei P. Balashov, Eleonora S. Imasheva, Rajni Govindjee, and Thomas G. Ebrey
Dept. of Cell & Structural Biology, University of Illinois
506 Morrill Hall, 505 S. Goodwin Ave. Urbana, IL 61801
Phone: (217) 333-2015, Fax: (217) 244-6615
E-mail: tebrey@ux1.cso.uiuc.edu
TITRATION OF ASPARTATE-85 IN BACTERIORHODOPSIN: WHAT IT SAYS ABOUT
CHROMOPHORE ISOMERIZATION AND PROTON RELEASE
ABSTRACT
Titration of Asp-85, the proton acceptor and part of the counterion
in bacteriorhodopsin over a wide pH range (between 2 and 11) leads
us to the following conclusions: 1) Asp-85 has a complex titration
curve with two pKas; in addition to a main transition with pKa =
2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150
mM KCl). This complex titration behavior of Asp-85 is explained by
interaction of Asp-85 with an ionizable residue X'. As follows from
the fit of the titration curve of Asp-85, deprotonation of X' increases
the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5.
Conversely protonation of Asp-85 decreases the pKa of X' by 4.9 units,
from 9.7 to 4.8. The interaction between Asp-85 and X' probably has
important implications for the mechanism of proton transfer. In the
photocycle after the formation of M intermediate (and protonation
of Asp-85) the group X' should release a proton. This deprotonated
state of X' would stabilize the protonated state of Asp-85. 2) Thermal
isomerization of the chromophore (dark adaptation) occurs upon
transient protonation of Asp-85 and formation of the blue membrane.
The latter conclusion is based on the observation that the rate
constant of dark adaptation is directly proportional to the fraction
of blue membrane (in which Asp-85 is protonated) between pH 2 and 11.
The rate constant of isomerization is at least 104 times faster in the
blue membrane than in the purple membrane. The protonated state of
Asp-85 probably is important for the catalysis of not only all-trans
<=> 13-cis thermal isomerization during dark adaptation but also
of the reisomerization of the chromophore from 13-cis to all-trans
configuration during N -> O -> bR transition in the photocycle. This
would explain why Asp-85 stays protonated in the N and O intermediates.
Copyright 1996 by the Biophysical Society
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