Biophys J, November 1999, p. 2750-2763, Vol. 77, No. 5

Two Groups Control Light-Induced Schiff Base Deprotonation and the Proton Affinity of Asp⁸⁵ in the Arg⁸²His Mutant of Bacteriorhodopsin

 ^*Center for Biophysics and Computational Biology and Departments of Cell and Structural Biology and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and  ^#Medical University of South Carolina, Charleston, South Carolina 29425 USA

Arg⁸² is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg⁸² controls the pK_as of Asp⁸⁵ and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg⁸² in light-induced proton pumping, we replaced Arg⁸² with histidine and studied the resulting pigment and its photochemical properties. The main pK_a of the purple-to-blue transition (pK_a of Asp⁸⁵) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK_a 4.5 to a value ~40% of that in the WT. A transition with a similar pK_a is observed in the pH dependence of the rate constant of dark adaptation, k_da. These data can be explained, assuming that some group deprotonates with pK_a 4.5, causing an increase in the pK_a of Asp⁸⁵ and thus affecting k_da and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 µs to 75 µs with pK_a 9.4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK_a. We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp⁸⁵ and the proton release group is weakened in R82H. The pK_a of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O  bR transition with   45 ms. This transition is slowed in R82H, indicating that Arg⁸² is important for the proton transfer from Asp⁸⁵ to the proton release group. A model describing the interaction of Asp⁸⁵ with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Biophys J, November 1999, p. 2750-2763, Vol. 77, No. 5
© 1999 by the Biophysical Society   0006-3495/99/11/2750/14  $2.00

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