The individual resonance Raman spectra of the PM568 and M412 forms of light-adapted purple membrane from Halobacterium halobium have been measured using the newly developed flow technique. For comparison purposes, the Raman spectra of the model chromophores, all-trans- and 13-cis retinal n-butylamine, both as protonated and unprotonated Schiff bases, have also been obtained. In agreement with previous work, the Raman data indicate that the retinal chromophore is linked to the purple membrane protein via a protonated. Schiff base in the case of the PM568 and an unprotonated Schiff base for the M412 form. The basic mechanism for color regulation in both forms appears to be electron delocalization. The spectral features of the two forms are different from each other and different from the model compound spectra.

The resonance Raman spectra of bovine metarhodopsin I and metarhodopsin II have been measured. The spectra are compared with model chromophore resonance Raman data. It was found that metarhodopsin I is linked to opsin via a protonated Schiff base linkage, whereas metarhodopsin II is linked by an unprotonated Schiff base. A recent suggestion that the chromophore of metarhodopsin II is retinal is explicitly disproved. The chromophores of both metarhodopsins are found to have an essentially all-trans conformation. The basic mechanism for color regulation in both forms appears to be electron delocalization. The data tend to support the model of cis-trans isomerization as the primary mechanism for vision. Also, the conclusions and inferences of this work on energy uses and storage by rhodopsin in neural generation are discussed.

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin have been obtained at low temperature. Application of the double beam "pump-probe" technique allows us to extract a complete bathorhodopsin spectrum from the mixture in both protonated and deuterated media. Our results show that the Schiff base of bathorhodopsin is fully protonated and that the extent of protonation is unaffected by its photochemical formation from either rhodopsin or isorhodopsin. The Raman spectrum of bathorhodopsin is significantly different than that of either parent pigment, thus supporting the notion that a geometric change in the chromophore is an important component of the primary photochemical event in vision. A normal mode analysis is carried out with particular attention devoted to the factors that determine the frequency of the C=N stretching vibration. We find that the increased frequency of this mode in protonated relative to unprotonated Schiff bases is due to coupling between C=N stretching and C=N-H bending motions, and the shift observed upon deuteration of the Schiff base can also be understood in these terms. Various models for the primary event are discussed in light of our experimental and theoretical results.

No abstract