Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp(85) in the Arg(82) his mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding p ocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) control s the pK(a)s of Asp(85) and the proton release group and is essential for f ast light-induced proton release. To further investigate the role of Arg(82 ) in light-induced proton pumping, we replaced Arg(82) with histidine and s tudied the resulting pigment and its photochemical properties. The main pK( a) of the purple-to-blue transition (pK(a) of Asp(85)) 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 de protonation (formation of the M intermediate) is observed. As the pH is inc reased from 3 to 7 the M yield increases with pK(a) 4.5 to a value similar to 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 dat a can be explained, assuming that some group deprotonates with pK(a) 4.5, c ausing an increase in the pK(a) of Asp(85) 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-f old increase in the yield of M and a decrease in its rise time from 200 mu s to 75 mu s 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 re lease group deprotonates in the unphotolyzed pigment, causing a transformat ion of the pigment into a red-shifted "alkaline" form which has a faster ra te of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versu s 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximate to 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp (85) to the proton release group. A model describing the interaction of Asp (85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.