Factors affecting the formation of an M-like intermediate in the photocycle of 13-cis-bacteriorhodopsin.

The light-induced proton pumping activity of bacteriorhodopsin (bR) is based on the photocycle of its light-adapted all-trans-retinal protein pigment. The photocycle of the 13-cis pigment lacks the M intermediate (which carries a deprotonated retinal Schiff base, characteristic of the all-trans photocycle) and is not associated with proton release and uptake. Aiming at establishing the reasons for the lack of light-induced Schiff base deprotonation and proton pumping in 13-cis-bR, we carried out pulsed-laser and continuous excitation experiments with artificial 13-cis-bR pigments derived from 13-demethylretinal, 13-demethyl-14-fluoro-bR, and 13-demethyl-12,14-difluoro-bR. Pulsed-laser photolysis shows that both M formation and proton pumping are restored in 13-cis-13-demethyl-bR by raising the pH to 8.5-9. M formation, but not proton pumping, is restored at neutral pH by 14-fluorine substitution. Continuous-illumination experiments lead, in all cases, to the generation of extremely long-lived (minutes to hours) M photoproducts. We show that such species are due to secondary photoreactions of late intermediates of the primary photolysis. Feasible mechanisms accounting for Schiff base deprotonation in the all-trans photocycle, but not in that of 13-cis-bR, are considered. Our findings favor a mechanism which attributes the lack of light-induced Schiff base deprotonation of 13-cis-bR to an insufficient change in the relative pKa of the donor (Schiff base) and acceptor (probably Asp-85) groups and/or to a high activation barrier for the proton transfer. The required change in relative pKas may be achieved either by deprotonation of a protein moiety (YH) with pKa approximately 8.5 or by fluorine substitution at position 14. Similarly, both YH titration and 14-fluorine substitution may reduce the barrier for proton transfer by affecting H-bonding interactions in the vicinity of the Schiff base linkage. The lack of proton release and uptake in the photocycle of 13-cis-13-demethyl-14-fluoro-bR, despite the presence of an M intermediate, is discussed. It appears that Schiff base deprotonation does not essentially imply proton release and uptake. Our conclusions bear on the molecular mechanism of the photocycle and of proton pumping in all-trans-bR.