A Role for Internal Water Molecules in Proton Affinity Changes in the Schiff Base and Asp85 for One-way Proton Transfer in Bacteriorhodopsin[dagger]

Photochemistry and Photobiology, Jul/Aug 2008 by Morgan, Joel E, Gennis, Robert B, Maeda, Akio

An FTfR study of L at low temperature has shown that the stretching vibration of the C^sub 14^-C^sub 15^-bond is located at 1155 cm^sup -1^ (46), a frequency lower than the corresponding hand observed at 1172 cm^sup -1^ in a resonance Raman study at room temperature (47). However, the observed frequency of 1155 cm^sup -1^ is far above that of the conformationally locked, authentic, 14-s-cis Schiff base of 12-N-ethano retinal (48), at 1096 cm^sup -1^, indicating that the C^sub 14^-C^sub 15^-bond of L has a conformation close to trans. Distortion of the Schiff base that was suggested by other studies (49,50) was not detected in the models of 1cuq (25) and 1r3p (28), which show full rotation of the C^sub 13^ = C^sub 14^ bond without other changes. This 1155 cm^sup -1^ band, due to the C^sub 14^-C^sub 15^ stretching vibration, is located at the same frequency in D^sub 2^O as in H2O (46). indicating that the C = N configuration is trans on the basis of the proposal in Smith et al. (51). in contrast, a substantial increase in the frequency might have been expected for the half-way rotation of the C=N bond shown in 2ntwc, the structure on which the proposed orientation of the Schiff base toward the extracellular side was largely based (29). As judged from the 13-cis, 14-s-trans, 15-trans configuration deduced from these vibrational studies, even with distortion around C^sub 15^, the N-H bond of L may be oriented to the cytoplasmic side. However, these arguments on the vibrational results should be further examined by measuring the distances between relevant atoms in solid state NMR studies like that applied to the C^sub 14^-C^sub 15^ bond in M (52).

On the other hand, L at room temperature seems to have an undistorted conformation around the Schiff base. Resonance Raman spectra of L at room temperature show that the chromophore has the con figuration 13-cis, 14-s-Trans, 15-trans (47,51). L in the time-resolved FTIR study did not exhibit the 1155 cm^sup -1^ band (see fig. 5 in Morgan et al. [I]). suggesting that there is no distortion in the C^sub 14^-C^sub 15^ bond.

The complex structure of the Schiff base interacting with the rigid protein, as suggested by neutron diffraction measurements (53), does not allow large amplitude motions. In the L intermediate, produced at 170 K, this structural rigidity may block a structural change, which would involve the relocation of the water cluster toward the site it characteristically occupies in M. Time-resolved visible spcctroscopy has revealed two consecutive L states, L^sub 1^ and L^sub 2^ (54). The visible spectrum of L^sub 1^, which arises in the sub-microsecond time domain before the appearance of normal L (presumably L^sup 2^ from the time constant), shows a more substantial tail toward longer wavelengths, like L at low temperature (30), probably due to some distortion around the Schiff base. The FTIR spectrum in a time range similar to that for L^sub 1^ shows changes in the bands due to the chromophore and Asp96/Asp115 similar to those in L, which occur in advance of the Schiff base changes observed in normal L (55). However, this precursor of L did not show changes in vibrational bands characteristic of those of L at low temperatures: the Schiff base bands at 1310 and 1075/1064/1056 cm^sup -1^.