Water Structural Changes at the Proton Uptake Site (the Thr46-Asp96 Domain) in the L Intermediate of Bacteriorhodopsin

• Published in: Biochemistry (Easton) Vol. 34; no. 21; pp. 7088 - 7093
• Main Authors: Yamazaki, Yoichi, Hatanaka, Minoru, Kandori, Hideki, Sasaki, Jun, Karstens, Willem F. Jan, Raap, Jan, Lugtenburg, Johan, Bizounok, Marina, Herzfeld, Judith
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.05.1995
• Subjects: Aspartic Acid - chemistry; Bacteriorhodopsins - chemistry; Hydrogen Bonding; Light; Oxygen Isotopes; Protein Conformation; Protons; Schiff Bases - chemistry; Spectroscopy, Fourier Transform Infrared; Threonine - chemistry; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00021a021

Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm-1, change into stronger H-bonding states in L of the wild type. Thr46-->Val substitution abolished these bands in spite of the fact that [3-18O]threonine-labeling did not shift them, indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96-->Asn substitution in Thr46-->Val/Asp96-->Asn. A single Asp96-->Asn substitution abolished the 3607 cm-1 band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two vibrational bands of the Schiff base mode at 1312 and 1301 cm-1 and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base with Asp85, that is far removed from this region.