title: Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M----N transition.
abstract: The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96----Ala and Asp-96----Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96----Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L----M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N----O or O----bR transition.
authors.affiliation: Physics Department, Boston University, Massachusetts 02215.
authors.forename: O
authors.initials: O
authors.lastname: Bousché
authors.forename: M
authors.initials: M
authors.lastname: Braiman
authors.forename: Y W
authors.initials: YW
authors.lastname: He
authors.forename: T
authors.initials: T
authors.lastname: Marti
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D005583:Fourier Analysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012545:Schiff Bases; D013055:Spectrophotometry, Infrared
chemical_list: D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins