In the photocycle of bacteriorhodopsin (BR), the first proton movement, from the Schiff base to Asp85, occurs after the formation of the L intermediate. In L, the C=N bond of the Schiff base is strained, and the nitrogen interacts strongly with its counterion. The present study seeks to detect the interaction of internal water molecules with the Schiff base in L using difference FTIR spectroscopy at 170 K. The coupled modes of the hydrogen-out-of plane bending vibrations (HOOPs) of the N-H and C15-H of the protonated Schiff base are detected as a broad band centered at 911 cm-1 for BR. A set of bands at 1073, 1064, and 1056 cm-1 for L is shown to arise from the coupling of the HOOP with the overtones of interacting water O-H vibrations. Interaction with water was shown by the decreased intensity of the HOOPs of L in H218O and by the influence of mutants that have been shown to perturb specific internal water molecules in BR. In contrast, the HOOP band of initial BR was not affected by these mutations. In D85N, the coupled HOOP of BR is depleted, while the coupled HOOPs of L are shifted. The results indicate that the Schiff base interacts with water in the L state but in a different manner than in the BR state. Moreover, the effects of mutations suggest that cytoplasmic water close to Thr46 (Wat46) either interacts stronger with the Schiff base in L or that it is important in stabilizing another water that does.
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