Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released a t the extracellular surface of the purple membrane. Structural studies as w ell as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. The se 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 i mportant residue is R134: it is located close to E194 and might interact el ectrostatically to affect the pK(a) of E194 and light-induced proton releas e. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transition s associated with proton transfer. By measuring the light-induced absorptio n changes versus pH, we found that the R134H mutation results in an increas e 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, release s the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowin g of the rate of the O --> bR transition at low pH and probably is the caus e 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 faction of O increases greatly upon decreasing the pH. These results provide furthe r support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also prov ide further evidence for the importance of the R134-E194 interaction in mod ulating proton release from D85 after light has led to its being protonated .