Lowering the intrinsic pKa of the chromophore's Schiff base can restore its light-induced deprotonation in the inactive Tyr-57–>Asn mutant of bacteriorhodopsin.

Following light absorption, at neutral pH the bacteriorhodopsin mutant Y57N does not show Schiff base deprotonation (no M intermediate) or proton pumping activity. We reasoned that this might be due to improper delta pKa between the proton-donating Schiff base and the proton-accepting Asp-85 after light absorption. To test this, we reduced the intrinsic pKa of the protonated Schiff base in the pigment (and thus in the photointermediates) by replacing the retinal chromophore with an analogue, 14-F retinal. This substitution restores light-induced M formation, strongly suggesting that light-induced Schiff base deprotonation is accomplished by lowering its pKa during the photochemical cycle. Thus, while it is generally accepted that the Schiff base deprotonation during the photocycle takes place because of the light-induced reduction in its pKa, we provide here the first experimental evidence of this phenomenon.