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 pK(a) of the latter group and results in its protonation.
|Original language||English (US)|
|Number of pages||11|
|Journal||Proteins: Structure, Function and Genetics|
|State||Published - 1988|
ASJC Scopus subject areas
- Structural Biology