TO explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pK(a) of about 1.7 pH units lower than that of Arg, In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pK(a) of the purple-to-blue transition at low pH (which reflects the pK;, of Asp85) is 3.6 +/- 0.1, At high pH a second inflection in the blue-to-purple transition with pK(a) = 8.0 is found. The complex titration behavior of Asp85 indicates that the pK, of Asp85 depends on the protonation state of another amino acid residue, X', which has a pK(a) = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pK(a) of Asp85 from 3.7 to 6.0, In turn, protonation of Asp85 decreases the pK(a) of X' by 2.3 pH units, This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, k(da), is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85, The pH dependence of k(da) shows that two groups with pK(a1) = 3.9 and pK(a2) = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pK(a2) in R82K compared to the wild type (WT) (pK(a2) = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or:lt least that these groups are the principal part of a cluster of residues that constitute X'), (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT, A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released. fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arl in this process, (4) R82K shows a pH-independent rate of M formation, lack of accumulation of the O intermediate, and fast decay of the bathoproduct of the 13-cis pigment, K-c (0.2 ms), At low and neutral pH, R82K contains a significant fraction of 13-cis-bR in the light-adapted state since trans-bR is convened back into 13-cis-bR under illumination at pH < 9, The pH dependence of light adaptation results in a pi-I-dependent yield of the M intermediate (pK(a) = 8.0). Two other reversible transitions with similar pK(a)'s are observed: a 2 nm red shift of the chromophore absorption band and a red shift of the absorption band of a Trp residue, (5) Lys82 is accessible to modification by acetic anhydride. Acetylation of Lys82 shifts the pK(a) of Asp85 to 6.5, which agrees with the prediction of our model for the pK(a) of Asp85 when X' is deprotonated.
