LOWERING THE INTRINSIC PK(A) OF THE CHROMOPHORES SCHIFF-BASE CAN RESTORE ITS LIGHT-INDUCED DEPROTONATION IN THE INACTIVE TYR-57-]ASN MUTANTOF BACTERIORHODOPSIN

Following light absorption, at neutral pH the bacterio rhodopsin mutan t Y57N does not show Schiff base deprotonation (no M intermediate) or proton pumping activity. We reasoned that this might be due to imprope r Delta pK(a) between the proton-donating Schiff base and the proton-a ccepting Asp-85 after light absorption. To test this, we reduced the i ntrinsic pK(a) of the protonated Schiff base in the pigment (and thus in the photointermediates) by replacing the retinal chromophore with a n analogue, 14-F retinal. This substitution restores light-induced M f ormation, strongly suggesting that light-induced Schiff base deprotona tion is accomplished by lowering its pK(a) during the photochemical cy cle. Thus, while it is generally accepted that the Schiff base deproto nation during the photocycle takes place because of the light-induced reduction in its pK(a), we provide here the first experimental evidenc e of this phenomenon.