Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

Mark S Braiman, T. Mogi, L. J. Stern, N. R. Hackett, B. H. Chao, H. G. Khorana, K. J. Rothschild

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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 pK(a) of the latter group and results in its protonation.

Original languageEnglish (US)
Pages (from-to)219-229
Number of pages11
JournalProteins: Structure, Function and Genetics
Volume3
Issue number4
StatePublished - 1988
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology

Cite this

Braiman, M. S., Mogi, T., Stern, L. J., Hackett, N. R., Chao, B. H., Khorana, H. G., & Rothschild, K. J. (1988). Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle. Proteins: Structure, Function and Genetics, 3(4), 219-229.