Investigation of the Primary Photochemistry of Bacteriorhodopsin by Low‐Temperature Fourier‐Transform Infrared Spectroscopy

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Abstract
The method of Fourrier‐transform infrared difference spectroscopy was applied to investigate the transition at 77K of bacteriorhodopsin in its light‐adapted form to K610, the first intermediate which is stable at low temperature. In addition to unmodified bacteriorhodopsin, bacteriorhodopsin in 2H2O and bacteriorhodopsin containing [15‐2H]retinal was used. The results show that major rearrangements occur in the Schiff base in this transition. It is not possible to identify a C=N stretching vibration of the Schiff base in K610. The identification of an N‐H bending vibration in K610 shows that the nitrogen of the previous Schiff base still has a proton attached. The fingerprint region exhibits very unusual features for K610 and bears no similarity to protonated retinylidene Schiff base model compounds of any isomeric composition. Therefore, no conclusions on the isomeric state of the retinal in K610 can be drawn. The spectra show that the terminal part of the retinal is predominantly reflected in the difference spectra. This indicates that the most polar part of the retinal is located near the Schiff base. We have evidence for protein molecular changes occurring in this transition at 77K.