Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base
- Freie Universitaet Berlin (Germany, F.R.)
Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-((3-cholamidopropyl)dimethylammonio)-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base.
- OSTI ID:
- 7192258
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Journal Issue: 3 Vol. 87:3; ISSN PNASA; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
Similar Records
A linkage of the pK{sub a}`s of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin
Uv-visible spectroscopy of bacteriorhodopsin mutants: Substitution of Arg-82, Asp-85, Tyr-185, and Asp-212 results in abnormal light-dark adaptation
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTRA
AMINO ACIDS
ARGININE
ASPARTIC ACID
BACTERIA
BARYONS
CARBOXYLIC ACIDS
CLONING
ELEMENTARY PARTICLES
ESCHERICHIA COLI
FERMIONS
GENES
HADRONS
IMINES
MICROORGANISMS
MUTATIONS
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
PROTEINS
PROTONS
RHODOPSIN
SCHIFF BASES
SPECTRA
SPECTROPHOTOMETRY
TRANSLOCATION