Glutamic acid 204 is the terminal proton release group at the extracellular surface of bacteriorhodopsin.
We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C = O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm-1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm-1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm-1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.
Brown
L S
LS
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Sasaki
J
J
Kandori
H
H
Maeda
A
A
Needleman
R
R
Lanyi
J K
JK
D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006217:Halobacterium / Q000737:chemistry / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D015394:Molecular Structure; D010777:Photochemistry; D017354:Point Mutation; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry
D011522:Protons; D012545:Schiff Bases; D014867:Water; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins