Biophysical Journal
August 1996
Arginine-82 Regulates the pKa of the Group Responsible for the Light-driven Proton Release in Bacteriorhodopsin
Rajni Govindjee, Saurav Misra, Sergei P. Balashov, Thomas G. Ebrey,
Rosalie K. Crouch *, 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
In wild type (WT) bacteriorhodopsin (bR) light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants where R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q we find that the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are: (1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pHs, suggesting that the proton release group has not been eliminated. (2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the WT (in 150 mM salt) the pKa of this group decreases from ca. 9.5 in the unphotolyzed pigment to ca. 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants, the respective pKa's of the proton release group in the unphotolyzed pigment and in M are ca. 8 and 7.5 in R82Q (in 1 M salt), and ca. 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. (3) Early proton release in R82Q can be detected as a photocurrent signal which is kinetically distinct from those photocurrents due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M -> N transition. (4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O -> bR transition, probably from D85 since the normal proton release group cannot deprotonate at this pH. (5) The time constant of early proton release is increased from 85 µs in the WT to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. (6) In the E204Q mutant only late proton release is observed both at neutral and alkaline pH, consistent with E204 being the proton release group as has been proposed by Brown et al. (1995a). The proton release is concurrent with the O-> bR transition, as in R82Q at neutral pH.
Received for publication 14 February 1996 and in final form 25 April 1996.
Address reprint requests to Dr. Thomas G. Ebrey, Department of Cell & Structural Biology, University of Illinois, 506 Morrill Hall, 505 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-2015; Fax: 217-244-6615; tebrey@ux1.cso.uiuc.edu
Copyright 1996 by the Biophysical Society
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