A Role for Internal Water Molecules in Proton Affinity Changes in the Schiff Base and Asp85 for One-way Proton Transfer in Bacteriorhodopsin[dagger]

Photochemistry and Photobiology, Jul/Aug 2008 by Morgan, Joel E, Gennis, Robert B, Maeda, Akio

Titration studies on the BR state show that deprotonation of the PRG causes the pK^sub a^ of Asp85 to increase to around 7 (5,59). In contrast, other observations of the properties of M show that Asp85 remains prolonaled even at pH 10 (49,60,61). The pK^sub a^ of Asp85 may be much higher than that of the Schiff base of N, 8.2 (62), preventing the reverse flow of the proton of Asp85 to the Schiff base. Below we will discuss several factors that may stabilize the prolonated Asp85.

THE ROLE OF ASP212 IN INCREASING THE PK^sub A^ OF ASP85 IN M

Asp212 and Arg82 are the nearest charged residues to Asp85. The spatial relationship of these residues in BR is shown in Fig. 1. D212N is inactive in proton pumping even though it undergoes the L to N transition in the photocycle (63,64). Proton pumping in D212N can be partially restored by the addition of chloride to the sample, though only below pH 7 (65,66), and also by a second mutation that restores the charge balance in D212N/R82Q (67). The photocycle of D212N/R82Q proceeds from L to N without apparent accumulation of M, but replacement of the retinal by 14-fluoro retinal, to decrease the pK^sub a^ of the Schiff base, restores the M formation. A lowered proton affinity of Asp85 in this mutant may be responsible for the apparent absence of M. Thus, the involvement of Asp212 in increasing the pK^sub a^ of Asp85 in the photocycle is suggested.

Spectroscopic data showing more rapid formation of M in the mutants of Arg82 (68,69), and the absence of M in E194Q (70) both suggest that the increased pK^sub a^ of Asp85 in M is brought about by disruption of the interaction between Asp212 and Arg82 under the influence of Glu 194, which attracts Arg82. Crystallographic models of M of wild type bacteriorhodopsin (see protein databank entries 1cwq [42], 1kg8 [71], 1m0m [6] and liw9 [43]) as well as the M intermediate structure for the E204Q mutant in 1f4z (41) show that the guanidium group of Arg82 moves away from Asp212 toward Glu194 in M, whereas a structure that has been claimed to be M^sub 1^ (obtained at acidic pH where the PRG remains protonated in M), 1p8h (39), retains contact between Arg82 and Asp212. Thus, Asp212, the Schiff base and the PRG are all expected to regulate the proton affinity of Asp85 in M. We propose that Asp212 breaks its interaction with Arg82 and the Schiff base in M. Removal of the positive charges of the Schiff base and Arg82 pushes the pK^sub a^ of the carboxylic acids higher. Asp212 forms a di-carboxylic acid pair together with Asp85, in which one of the pK^sub a^ values is elevated under influence of the other, as observed in malonic acid (2.8 and 5.8) compared to acetic acid (4.75) (72). These experimental results, however, are not consistent with an effect by which Asp212 would destabilize the protonated Asp85, as was concluded by QM/MM calculations based on the simple model including Asp85, Asp212, Thr89 and the Schiff base (7,8). The reason is unclear, but an active role of mobile water molecules is anticipated (see below).