Biochemistry
1999, 38(7); 2026-2039
The Proton Release Group of Bacteriorhodopsin Controls
the Rate of the Final Step of Its Photocycle at Low pH
Balashov, S. P., Lu, M., Imasheva, E. S., Govindjee, R.,
Ebrey, T. G.,
Othersen, B., III *, Chen, Y. *,
Crouch, R. K. *, Menick, D. R. *, and Donald R. Menick
*
Center for Biophysics and Computational Biology, and the
Department of Cell and Structural Biology,
University of Illinois at Urbana-Champaign, Urbana, IL
61801
* Medical University of South Carolina, Charleston,
South Carolina 29425
Abstract
The factors determining the pH dependence of the formation
and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle
were investigated in the wild type pigment and in mutants of Glu-194 and
Glu-204, key residues of the proton release group (PRG) in bR. We have
found that in the WT, the rate constant of O -> bR transition decreases
30 fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O
slows down the rise and decay of the O intermediate in the WT at pH 3.5
by a factor of 5.5. We suggest that the rate of the O to bR transition
(which reflects the rate of deprotonation of the primary proton acceptor
Asp-85) at low pH is controlled by the deprotonation of the proton release
group. To test this hypothesis, we studied the E194D mutant. We show that
the pKa of the proton release group in the ground state of the E194D mutant,
when Asp-85 is protonated, is increased by 1.2 pK units compared to WT.
We found a similar increase in the pKa of the rate constant of the O to
bR transition in E194D. This provides further evidence that the rate of
the O to bR transition is controlled by the proton release group. In a
further test the E194Q mutation, which disables the PRG and slows down
proton release, almost completely eliminates the pH dependence of O decay
at pHs below 6. A second phenomenon we investigated was that in the WT
at neutral and alkaline pH, the fraction of the O intermediate decreases
with pKa 7.5. A similar pH dependence is observed in mutants in which the
PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction
of the O intermediate with pKa around 7.5 is not controlled by the proton
release group. We propose that the group with pKa 7.5 is Asp-96. Slowing
down of reprotonation of Asp-96 at high pH is the cause of the decrease
in the rate of N -> O transition, leading to the decrease in the fraction
of O.
Received for publication 10 August 1998 and in final form 17 November
1998.
Address reprint requests to Dr. Sergei P. Balashov, Department
of Cell & Structural Biology, University of Illinois, 506 Morrill Hall,
505 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-2435; Fax: 217-244-6615;
sbalasho@uiuc.edu
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