A LINKAGE OF THE PK(A)S OF ASP-85 AND GLU-204 FORMS PART OF THE REPROTONATION SWITCH OF BACTERIORHODOPSIN

Because asp-85 is the acceptor of the retinal Schiff base proton durin g light-driven proton transport by bacteriorhodopsin, modulation of it s pK(a) in the photocycle is to be expected, The complex titration of asp-85 in the unphotolyzed protein was suggested [Balashov, S. P., Gov indjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch , R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] to reflect the dependence of this residue on the protonation state of another, un identified group, From the pH dependencies of the rate constant for th e thermal equilibration of retinal isomeric states (dark adaptation) a nd the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. T he nature of its interaction with asp-85 is that at neutral pH either residue can be anionic but not both. This is consistent with our recen t finding that glu-204 is the origin of the proton released to the ext racellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the S chiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface. The deprot onation of glu-204, in turn, raises the pK, of asp-85, and the equilib rium with the Schiff base shifts toward complete proton transfer. This constitutes: the first phase of the reprotonation switch because it e xcludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the chan ge of the access of the Schiff base toward the cytoplasmic side as the second phase of the switch, and the lowering the pK, of asp-96, so as to make it a proton donor, as the third phase.