ESTIMATED ACID DISSOCIATION-CONSTANTS OF THE SCHIFF-BASE, ASP-85, ANDARG-82 DURING THE BACTERIORHODOPSIN PHOTOCYCLE

The pK(a) values of D85 in the wild-type and R82Q, as well as R82A rec ombinant bacteriorhodopsins, and the Schiff base in the D85N, D85T, an d D85N/R82Q proteins, have been determined by spectroscopic titrations in the dark. They are used to estimate the coulombic interaction ener gies and the pK(a) values of the Schiff base, D85, and R82 during prot on transfer from the Schiff base to D85, and the subsequent proton rel ease to the bulk in the initial part of the photocycle. The pK(a) of t he Schiff base before photoexcitation is calculated to be in effect on ly 5.3-5.7 pH units higher than that of D85; overcoming this to allow proton transfer to D85 requires about two thirds of the estimated exce ss free energy retained after absorption of a photon. The proton relea se on the extracellular surface is from an unidentified residue whose pK(a) is lowered to about 6 after deprotonation of the Schiff base (Zi manyi, L., G. Varo, M. Chang, B. Ni, R. Needleman, and J. K. Lanyi. 19 92. Biochemistry. 31:8535-8543). We calculate that the pK(a) of R82 is 13.8 before photoexcitation, and it is lowered after proton exchange between the Schiff base and D85 only by 1.5-2.3 pH units. Therefore, c oulombic interactions alone do not appear to change the pK(a) of R82 a s much as required if it were the proton release group.