We used polarized Fourier transform infrared (FTIR) spectroscopy to investigate the structural change of bacteriorhodopsin (BR) upon photoisomerization of the retinal chromophore. By measuring the difference spectra between the K-intermediate and BR in the whole mid-infrared region (700−4000 cm-1) at 77 K, complete vibrational information was obtained on how the protein responds to the displacement of the chromophore. In particular, changes in O−H and N−H stretching vibrations, which directly probe the hydrogen bonding strength, have provided not only the relevant frequencies but also their angles to the membrane normal. Structural perturbation of the peptide backbone appears in the 3270−3320 cm-1 (peptide N−H stretch) and the 1650−1670 cm-1 (peptide CO stretch) regions. These peptide bands are insensitive to H−D exchange, and the dipole moments of the N−H and CO stretches are parallel to the membrane normal. In contrast, several bands are downshifted upon D2O substitution, indicating that O−H or N−H groups that participate in a hydrogen bonding network near the chromophore change upon cis−trans isomerization.

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 18(O)-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.

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The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtoseconds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein changes on photoisomerization by using the O-H groups of threonines as internal probes. Polarized Fourier-transform IR spectroscopy of [3-18 O]threonine-labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D 2O, but two are not. A comprehensive mutation study indicates that the residues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the structural change of Thr-17, which is located >11 Å from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion. B acteriorhodopsin (BR) is a light-driven proton pump inHalobacterium salinarum that contains all-trans retinal as chromophore (1-4). Its tertiary structure has been determined recently by cryoelectron microscopy (5-7) and x-ray crystallography (8-13). The retinal binds covalently to Lys-216 through a protonated Schiff base linkage. Absorption of light triggers a cyclic reaction that comprises a series of intermediates, designated as the J, K, KL, L, M, N, and O states (1-4). Protein structural changes in these intermediate states cause proton translocation across the protein, and their mechanism is the central question in current studies of BR (14).The all-trans to 13-cis photoisomerization leads to the formation of the primary K intermediate (15)(16)(17). It is well known that photoisomerization in BR is highly selective and efficient. In solution, the photoproduct of all-trans retinal with protonated Schiff base is mainly 11-cis [82% (vol/vol) 11-cis͞6% (vol/vol) 13-cis͞12% (vol/vol) 9-cis in methanol; ref. 18], whereas in BR, the photoproduct is 100% (vol͞vol) 13-cis. The quantum efficiency for isomerization in BR (Ϸ0.6; refs. 19 and 20) is much higher than for retinal in solution (0.13 in methanol; ref. 18). This efficiency is correlated closely with the rate constant of the isomerization, because it occurs on the femtosecond time scale. The protein environment thus certainly facilitates the specificity of the reaction of the retinal chromophore.How does the highly selective and efficient isomerization occur in BR? How does the protein respond to the chromophore motion? To address these questions, structural analysis of the primary intermediates is essential. Cryoelectron microscopy of the K intermediate reported that the structural change at this stage is not resolved with 3.5-Å resolution (21). More recent analysis of the K intermediate by x-ray crystallography identified few...