Abstract
The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O−H and N−H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899−7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm-1 in bacteriorhodopsin and 2466 cm-1 in the K intermediate, in D2O) exhibited 18O-induced isotope shift. The O−H stretching vibrations of the threonine side chain correspond to 3378 cm-1 in bacteriorhodopsin and to 3317 cm-1 in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O−H stretch frequency of neat secondary alcohol is 3340−3355 cm-1. The O−H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O−H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S−H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828−5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.