Coupling of the reisomerization of the retinal, proton uptake, and reprotonation of Asp-96 in the N photointermediate of bacteriorhodopsin

In the N to O reaction of the bacteriorhodopsin photocycle, Asp-96 is proto nated from the cytoplasmic surface, and coupled to this, the retinal isomer izes from 13-cis,15-anti back to the initial all-trans configuration. To di ssect the two steps, and to better understand how and why they occur, we de scribe the properties of two groups of site-specific mutants in which the N intermediate has greatly increased lifetime. In the first group, with the mutations near the retinal, an unusual N state is produced in which the ret inal is 13-cis,15-anti but Asp-96 has a protonated carboxyl group. The appa rent pK(a) for the protonation is 7.5, as in the wild-type. It is likely th at here the interference with N decay is the result of steric conflict of s ide-chains with the retinal or with the side-chain of Lys-216 connected to the retinal, which delays the reisomerization after protonation of Asp-96. In the second group, with the mutations located near Asp-96 or between Asp- 96 and the cytoplasmic surface, reprotonation of Asp-96 is strongly perturb ed. The reisomerization of the retinal occurs only after recovery from a lo ng-living protein conformation in which reprotonation of Asp-96 is either e ntirely blocked or blocked at low pH.