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 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.
Dioumaev
A K
AK
Department of Physiology & Biophysics, University of California, Irvine, California 92697-4056, USA.
Richter
H T
HT
Brown
L S
LS
Tanio
M
M
Tuzi
S
S
Saito
H
H
Kimura
Y
Y
Needleman
R
R
Lanyi
J K
JK
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D019617:Halobacterium salinarum / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010777:Photochemistry; D017354:Point Mutation; D011487:Protein Conformation; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
D011522:Protons; D012545:Schiff Bases; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D012172:Retinaldehyde