Resonance Raman studies of the primary event

In the resonance Raman experiment, selectivity and sensitivity are achieved by the use of tunable lasers which allow the selection of frequencies in resonance with the electronic absorption of the retinal moiety. This permitted the observation of greatly enhanced scattering of the vibrational spectrum of this chromophore above the background of vibrations from the opsin matrix [130]. This technique has been found useful in the studies of a large number of biologically important molecules [131,132,229]. 

More recent work [136,137] confirmed the fully protonated state of the Schiff base linkage and reported additional features in the spectra of both squid and bovine rhodopsin. This led to the proposal of a distorted 1 l-c or 9-cis structure in bathorhodopsin and the conclusion that the primary event does not involve a cis to all -trans isomerization. The batho intermediate, however, could subsequently relax to an all -trans configuration by rotation around the C9-Cl0 and Cn-Cl2 bonds. 

The resonance Raman spectrum of bathorhodopsin has recently been analyzed in detail by using a two laser beam pump-probe technique [140-142], On the basis of new experimental data as well as theoretical calculations [142] of the C=N stretching and C=N—H bend frequencies and the large shift seen upon deuteration (25 cm 1), it was concluded that the proton in both rhodopsin and bathorhodopsin must be 

In a continuation of the above elegant work [144], the lines near 854, 875 and 922 cm-1, exclusively belonging to bathorhodopsin, were unambiguously assigned by the resonance Raman studies carried out on 10-deuterio- and 11,12-dideuterio-rhodopsins (1 and 2). Thus the 875 cm-1 line in bathorhodopsin was assigned to 

CI0H HOOP wag and the 922 cm-1 line to an HCn-Cl2H HOOP mode. The line at 854 cm-1 was attributed to the CI4H HOOP. As the intensities of the lines depend on the resonant excited state geometry, the enhancement of the 922 cm-1 line in bathorhodopsin indicates that the twist around the 11,12 bond is different in the excited state from that of the ground state, i.e., a situation expected to occur in cis-trans isomerization. Thus the resonance Raman data showed that the chromophore configuration in bathorhodopsin is a perturbed all-trans. 

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