Document Type

Article

Embargo Period

1-1-2024

Publication Date

11-1-1999

Abstract

Arg82 is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg82 controls the pKas 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 pKa of the purple-to-blue transition (pKa 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 pKa 4.5 to a value ~40% of that in the WT. A transition with a similar pKa is observed in the pH dependence of the rate constant of dark adaptation, kda. These data can be explained, assuming that some group deprotonates with pKa 4.5, causing an increase in the pKa of Asp85 and thus affecting kda 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 pKa 9.4. The chromophore absorption band undergoes a 4-nm red shift with a similar pKa. 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 pKa 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 τ ≈ 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.

Journal

Biophysical Journal

DOI

doi: 10.1016/s0006-3495(99)77108-0

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