title: Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay.
abstract: The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.
authors.affiliation: Physics Department, Boston University, MA 02215.
authors.forename: O
authors.initials: O
authors.lastname: Bousché
authors.forename: S
authors.initials: S
authors.lastname: Sonar
authors.forename: M P
authors.initials: MP
authors.lastname: Krebs
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011487:Protein Conformation; D011522:Protons; D013055:Spectrophotometry, Infrared
chemical_list: D011522:Protons; D001436:Bacteriorhodopsins