Time-resolved titrations of the Schiff base and of the Asp super(85) residue in artificial bacteriorhodopsins

Deprotonation/protonation processes involving the retinal Schiff base and the Asp super(85) residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pK sub(a) values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pK sub(a) values of the two moieties (12.2 plus or minus 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp super(85), respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pK sub(a) values of the above two residues are substantially modified: 13-CF sub(3) bR, pK sub(a) (Schiff base) = 8.2 plus or minus 0.2; 13-demethyl-11,14-epoxy bR, pK sub(a) (Schiff base) = 8.2 plus or minus 0.1 (in 0.1 M NaCl); aromatic bR, pK sub(a) (Asp super(85)) = 5.2 plus or minus 0.1 (in water). The R82Q bR mutant, pK sub(a) (Asp super(85)) approximately equal to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp super(85) moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7. The observed protonation rate constant in the pH = 7-9 range is k sub(a) = 6.0 x 10 super(7)/M/s, implying a reactivity that is lower by 3 orders of magnitude as compared to the diffusion-controlled rate constant of an elementary acid/base in homogeneous solutions. In the case of Asp super(85), k sub(a) could not be directly determined. The titration rates observed in the case of pigment IV are, however, consistent with a model in which the Schiff base and Asp super(85) are exposed to the extracellular side via the same proton channel. It is suggested that the rate-determining step in proton translocation via this channel is a transfer between Asp super(85) and the outside, rather than between Asp super(85) and the Schiff base. This conclusion applies independently