EXISTENCE OF A PROTON-TRANSFER CHAIN IN BACTERIORHODOPSIN - PARTICIPATION OF GLU-194 IN THE RELEASE OF PROTONS TO THE EXTRACELLULAR SURFACE

Glu-194 near the extracellular surface of bacteriorhodopsin is indispe nsable for proton release to the medium upon protanation of Asp-SS dur ing light-driven transport. As for Glu-204, its replacement with gluta mine (but not aspartate) abolishes both proton release and the anomalo us titration of Asp-85 that originates from coupling between the pK(a) of this buried aspartate and those of the other acidic groups, Unlike the case of Glu-204, however, replacement. of Glu-194 with aspartate raises the pK, for proton release. In Fourier transform infrared spect ra of the E194D mutant a prominent positive band is observed at 1720 c m(-1). It can be assigned from [4-C-13]aspartate and D2O isotope shift s to the C=O stretch of protonated Asp-194. Its rise correlates with p roton transfer from the retinal Schiff base to Asp-85, Its decay coinc ides with the appearance of a proton at the surface, detected under si milar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pK(a) of about 9. We show that this pK(a) is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation: wh ile C-13 NMR titration indicates that Asp-194 has an initial pK(a) of about 3. We propose that there is a chain of interacting residues betw een tile retinal Schiff base and the extracellular surface, After phot oisomerization of tile retinal the pK(a)'s change so as to allow (i) A sp-85 to become protonated by the Schiff base, (ii) the Glu-204 site t o transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the med ium.