Low Temperature FTIR Study of the Schiff Base Reprotonation During the M-to-bR Backphotoreaction: Asp 85 Reprotonates Two Distinct Types of Schiff Base Species at Different Temperatures

We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.