title: Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.
abstract: 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.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. kandori@photo2.biophys.kyoto-u.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Kandori
authors.forename: Y
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Y
authors.initials: Y
authors.lastname: Shichida
authors.forename: J
authors.initials: J
authors.lastname: Raap
authors.forename: J
authors.initials: J
authors.lastname: Lugtenburg
authors.forename: M
authors.initials: M
authors.lastname: Belenky
authors.forename: J
authors.initials: J
authors.lastname: Herzfeld
mesh_terms: 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*
chemical_list: D008565:Membrane Proteins; D010103:Oxygen Isotopes; D017494:Proton Pumps; D013912:Threonine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins