Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization

Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6873-7. doi: 10.1073/pnas.88.15.6873.

Abstract

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine*
  • Amino Acid Sequence
  • Bacteriorhodopsins / genetics
  • Bacteriorhodopsins / metabolism*
  • Cloning, Molecular
  • Darkness
  • Escherichia coli / genetics
  • Isomerism
  • Kinetics
  • Leucine*
  • Light
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protons
  • Recombinant Proteins / metabolism
  • Retinaldehyde / metabolism*
  • Rhodopsin / radiation effects
  • Spectrophotometry
  • Threonine*

Substances

  • Protons
  • Recombinant Proteins
  • Threonine
  • Bacteriorhodopsins
  • Rhodopsin
  • Leucine
  • Alanine
  • Retinaldehyde