Controlling the pK(a) of the bacteriorhodopsin Schiff base by use of artificial retinal analogues

Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF₃ substituent group were prepared. The effects of CF₃ on the spectra, photocycles, and Schiff base pK(a) values of the pigments were analyzed. A reduction of 5 units in the pK(a) of the Schiff base is observed when the CF₃ substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead (i) to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and (ii) to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M₄₁₂ intermediate) is determined by a structural change in the protein.