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 protei n pigment. The photocycle of the 13-cis pigment lacks the M intermedia te (which carries a deprotonated retinal Schiff base, characteristic o f the all-trans photocycle) and is not associated with proton release and uptake. Aiming at establishing the reasons for the lack of light-i nduced Schiff base deprotonation and proton pumping in 13-cis-bR, we c arried out pulsed-laser and continuous excitation experiments with art ificial 13-cis-bR pigments derived from 13-demethylretinal, 13-demethy l-14-fluoro-bR, and 13-demethyl-12,14-difluoro-bR. Pulsed-laser photol ysis 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 p roton pumping, is restored at neutral pH by 14-fluorine substitution. Continuous-illumination experiments lead, in all cases, to the generat ion of extremely long-lived (minutes to hours) M photoproducts. We sho w that such species are due to secondary photoreactions of late interm ediates 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 at tributes the lack of light-induced Schiff base deprotonation of 13-cis -bR to an insufficient change in the relative pK(a) of the donor (Schi ff base) and acceptor (probably Asp-85) groups and/or to a high activa tion barrier for the proton transfer. The required change in relative pK(a)s may be achieved either by deprotonation of a protein moiety (YH ) with pK(a) approximate to 8.5 or by fluorine substitution at positio n 14. Similarly, both YH titration and 14-fluorine substitution may re duce the barrier for proton transfer by affecting H-bonding interactio ns in the vicinity of the Schiff base linkage. The lack of proton rele ase and uptake in the photocycle of 13-cis-13-demethyl-14-fluoro-bR, d espite the presence of an M intermediate, is discussed. It appears tha t Schiff base deprotonation does not essentially imply proton release and uptake. Our conclusions bear on the molecular mechanism of the pho tocycle and of proton pumping in all-trans-bR.