THE RETINAL SCHIFF BASE-COUNTERION COMPLEX OF BACTERIORHODOPSIN - CHANGED GEOMETRY DURING THE PHOTOCYCLE IS A CAUSE OF PROTON-TRANSFER TO ASPARTATE-85

Bacteriorhodopsin contains all-trans-retinal linked via a protonated S chiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relatio nship of the Schiff base and D85 after the photoisomerization is a pos sible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that c ontact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimanyi, L., Va rp, G., Chang, M., Ni, B., Needleman, R., and Lanyi, J. K. (1992) Bioc hemistry 31, 8535-8543] we had described the kinetics of absorbance ch anges in the microsecond to millisecond time range after photoexcitati on with the scheme L double left right arrow M(1) double left right ar row M(2) + H+ (where the first equilibrium is the internal proton tran sfer and the second is proton release on the extracellular surface). T esting it at various pH values with mutants, where selected rate const ants are changed, now confirms the validity of this scheme. The kineti cs of the M state thus allowed examination of the transient equilibriu m that develops in the L double left right arrow M(1) reaction and rep resents the redistribution of the proton between the Schiff base and D 85. From the structure of the protein, the V49A and V49M residue repla cements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed. we found that they both changed the equilib rium toward the protonated Schiff base. In contrast, the residue repla cements A53V and A53G were predicted to move the Schiff base in opposi te directions, away from and closer to alignment with D85, respectivel y. The former indeed changed the equilibrium toward the protonated Sch iff base and the latter toward the deprotonated Schiff base. In additi on, the hydroxyl stretch band of a bound water in the L state was affe cted by all mutations that disfavor proton transfer to D85. We conclud e that the geometry of the proton donor and acceptor in the Schiff bas e-D85 pair, mediated by bound water, is a determinant of the proton tr ansfer equilibrium.