Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate.

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted Ol. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these Ol species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid−base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The Ol species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in Ol, Asp-85 is prot...