Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy

• Published in: Biochemistry (Easton) 1992-05, Vol.31 (19), p.4684-4690
• Main Authors: Maeda, Akio, Sasaki, Jun, Shichida, Yoshinori, Yoshizawa, Toru, Chang, Man, Ni, Baofu, Needleman, Richard, Lanyi, Janos K
• Format: Article
• Language: English
• Subjects: Acetates - chemistry; Acetic Acid; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; aspartic acid; Aspartic Acid - chemistry; bacteriorhodopsin; Bacteriorhodopsins - chemistry; Biological and medical sciences; Carbon Isotopes; characterization; Deuterium; Exobiology; Fourier Analysis; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Mutation; Non metallic chromoproteins, photoproteins; Proteins; Protons; Schiff Bases; Space life sciences; Spectrophotometry, Infrared; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00134a022

The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.