title: Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle.
abstract: The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.
authors.affiliation: Physics Department, Boston University, Massachusetts 02215.
authors.forename: M S
authors.initials: MS
authors.lastname: Braiman
authors.forename: T
authors.initials: T
authors.lastname: Mogi
authors.forename: L J
authors.initials: LJ
authors.lastname: Stern
authors.forename: N R
authors.initials: NR
authors.lastname: Hackett
authors.forename: B H
authors.initials: BH
authors.lastname: Chao
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics; D005583:Fourier Analysis; D006217:Halobacterium / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D011487:Protein Conformation; D011522:Protons; D013057:Spectrum Analysis; D014443:Tyrosine
chemical_list: D011522:Protons; D014443:Tyrosine; D001436:Bacteriorhodopsins