Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.
Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O--H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-(18)O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O--D group of Thr-89 in D(2)O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O--H (O--D) group of Thr-89 in M is indicated by a shift in the C==O stretching vibration of the former because of (18)O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.
Kandori
H
H
Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. kandori@photo2.biophys.kyoto-u.ac.jp
Yamazaki
Y
Y
Shichida
Y
Y
Raap
J
J
Lugtenburg
J
J
Belenky
M
M
Herzfeld
J
J
D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000737:chemistry*; D010103:Oxygen Isotopes; D017494:Proton Pumps / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*
D008565:Membrane Proteins; D010103:Oxygen Isotopes; D017494:Proton Pumps; D013912:Threonine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins