Generation of the O-630 photointermediate of bacteriorhodopsin is controlled by the state of protonation of several protein residues

The last stages of the photocycle of the photosynthetic pigment all-trans b acteriorhodopsin (bR(570)), as well as its proton pump mechanism, are marke dly pH dependent. We have measured the relative amount of the accumulated O -630 intermediate (Phi(r)), as well as its rise and decay rate constants (k (r) and k(d), respectively), over a wide pH range. The experiments were car ried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH d ependencies, s-shaped curves in the case of Phi(r) and bell-shaped curves f or k(r) and k(d), are interpreted in terms of the titration of three protei n residues denoted as R-1, R-2, and R-3. The R-1 titration is responsible f or the increase in Phi(r), k(r), and k(d) upon lowering the pH from pH appr oximate to 9.5 to 7. At low pH Phi(r) exhibits a secondary rise which is at tributed to the titration of a low pK(a) group, R-2. After reaching a maxim um at pH approximate to 7, k(r) and k(d) undergo a decrease upon decreasing the pH, which is attributed to the titration of R-3. All three titrations exhibit pK(a) values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) double left right arrow Blue (bR(605) ) equilibrium, divalent cations are substantially more effective than monov alent cations in shifting the pK(a) values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple double left right arrow Blue equilibrium, the salt binding si tes which control the pK(a) values of R-1, R-2, and R-3 are located on, or close to, the membrane surface. Possible identifications of the three prote in residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R-2 (Phi(r)) titration, suggesting that R-2 should be identified with Glu-204 or with a group whose pK(a) is affec ted by Glu-204. The relation between the R-1, R-2 and R-3 titrations and th e proton pump mechanism is discussed. It is evident that the pH dependence of Phi(r) is unrelated to the measured pK(a) of the group (XH) which releas es the proton to the extracellular medium during the photocycle. However, s ince the same residue may exhibit different pK(a) values at different stage s of the photocycle, it cannot be excluded that R-2 or R-3 may be identifie d with XH.