Role of aspartate-96 in proton translocation by bacteriorhodopsin.
Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1 of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.
Gerwert
K
K
Max-Planck-Institut für Ernährungsphysiologie, Dortmund, Federal Republic of Germany.
Hess
B
B
Soppa
J
J
Oesterhelt
D
D
D001216:Asparagine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000378:metabolism*; D005583:Fourier Analysis; D006217:Halobacterium / Q000378:metabolism; D011522:Protons; D013055:Spectrophotometry, Infrared
D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine