Mechanism of base-catalyzed Schiff base deprotonation in halorhodopsin

It has been shown earlier that the deprotonation of the Schiff base of illuminated halorhodopsin proceeds with a much lower pKa than that of the unilluminated pigment and the reversible protonation change is catalyzed by azide and cyanate [Hegemann, P., Oesterhelt, D., & Steiner, M. (1985) EMBO J. 4, 2347-2350]. We have studied the kinetics of the proton-transfer events with flash spectroscopy and compared a variety of anionic bases with different pKa with regard to their apparent binding constants and their catalytic activities. The results suggest a general base catalysis mechanism in which the anionic bases bind with apparently low affinity to halorhodopsin, although with some degree of size- and/or shape-dependent specificity. The locus of the catalysis is accessible from the cytoplasmic side of the membrane and is not at site I, where various anions bind and shift the pKa of the deprotonation. Neither is it at site II, where a few specific anions (like chloride) bind to the all-trans pigment. It may be concluded that while the all-trans pigment loses its Schiff base proton very rapidly at its pKa, there is a kinetic barrier to this deprotonation in the 13-cis photointermediate that can be partially overcome by the reversible protonation of an extrinsic anionic base, which shuttles protons between the interior of the protein and the aqueous medium. The need for an extrinsic proton acceptor for efficient deprotonation of the Schiff base of halorhodopsin is one of the main differences between this pigment and the analogous retinal protein, bacteriorhodopsin.