Spectroscopy and photochemistry of rhodopsin models: retinals, retinal Schiff bases and protonated retinal Schiff bases



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The n-butylamine Schiff bases of all-trans, 9-cis, 11-cis, and 13-cis retinal and the methylamine Schiff base of all-trans retinal have been prepared from the corresponding retinals. Detailed absorption spectral studies to ~48,000 kK at 77 and 295 K have been used to determine absorption maxima and oscillator strengths for each observed transition. Four absorption bands were observed at approximately 28,000, 33,000 to 39,000, 40,000 and 45,000 kK. Except for the first two bands of the Schiff bases being blue shifted about 1000 kK, the absorption spectra of retinals and their Schiff bases were very similar. The first absorption band in both retinals and Schiff bases was broad and unstructured, having an oscillator strength near unity, and was red shifted upon going from 295 to 77 K. The shift may be ascribed to a pure solvent effect except for the 11-cis retinal and Schiff base which underwent a geometry change upon cooling. The second observed band had vibrational components having an interband distance of 1200 to 1700 kK at 77 K. The third observed band in the Schiff bases was only slightly shifted from its corresponding retinal and had the same relative intensities; 11-cis > 13-cis > 9-cis > all-trans. Upon addition of HC1 gas to the a11-trans and 11-cis Schiff bases, the first band maxima was red shifted from -360 to -460 nm (~2800 kK), however, a peak could be detected at 542 nm at 77 K. At ~100 K this latter peak grew and the 460 peak decreased. The 542 nm band resulted from the interaction of HC1 along the polyene chain in addition to the protonation of the imine nitrogen. Protonation of the imine nitrogen resulted in a band maxima at ~450 nm. The fluorescence quantum yields, [Phi][lowered F], of the isomeric retinal Schiff bases and the protonated methyl and n-butyl Schiff bases of all-trans retinal have been determined at 77 K. No fluorescence was observed from any molecule at room temperature, [Phi][lowered F] <0.001. The emission band of the retinals and Schiff bases were similar, broad and unstructured, although the emission maxima was blue shifted ~20 nm in the Schiff bases. The dependence of [Phi][lowered F] upon the exciting wave-length was calculated from the fluorescence excitation spectrum of the all-trans, 9-cis and 13-cis retinals and their Schiff bases. While [Phi][lowered F] showed only a small dependence when exciting into the first absorption band of the Schiff bases or protonated Schiff bases, it demonstrated a large variation when exciting into the long wavelength absorption onset (400-440 nm) in the retinals. It is probable that the nature of the emitting state of the retinals is not the same as in the Schiff bases. Irradiation of the 9-cis, 11-cis, and 13-cis Schiff bases resulted in cis -> trans isomerization at 295 and 77 K for the Schiff bases. The trans cis -process occurred at 295 K but is quenched at 77 K. The activation energy for the 11-cis Schiff base isomerization was estimated to be -3 kcal/mole. The 11-cis Schiff base +HCl that had a band maxim at 542 nm underwent cis -> trans isomerization at 77 K.