Existence of a Proton Transfer Chain in Bacteriorhodopsin: Participation of Glu-194 in the Release of Protons to the Extracellular Surface

Citations Over TimeTop 10% of 1998 papers

Abstract

Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pKa 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 pKa for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm-1. It can be assigned from [4-13C]aspartate and D2O isotope shifts to the C&dbd;O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pKa of about 9. We show that this pKa is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation, while 13C NMR titration indicates that Asp-194 has an initial pKa of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pKa's change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to 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 medium.

Related Papers

• → Use of the fluorescent pH probe pyranine to detect heterogeneous directions of proton movement in bacteriorhodopsin reconstituted large liposomes(1985)57 cited
• → ON THE ROLE OF TYROSINE IN THE PHOTOCYCLE OF BACTERIORHODOPSIN(1982)31 cited
• → ON THE TYROSINATE INVOLVEMENT IN THE SCHIFF BASE DEPROTONATION IN THE PROTON PUMP CYCLE OF BACTERIORHODOPSIN(1984)15 cited
• → Time-resolved laser studies on the proton pump mechanism of bacteriorhodopsin(1991)
• → Time-resolved laser studies on the proton pump mechanism of bacteriorhodopsin. Progress report, January 31,1991--February 1, 1992(1991)