Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.