Vibrational Analysis of the all-trans-Retinal Chromophore in Light-Adapted Bacteriorhodopsin

Resonance Raman spectra of light-adapted bacteriorhodopsin (BR₅₆₈) have been obtained using purple membrane regenerated with isotopic retinal derivatives. The chromophore was labeled with^I3C at positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, while deuterium substitutions were made at positions 7, 8, 10, 11, 12, 14, and 15 and on the Schiff base nitrogen. On the basis of the observed isotopic shifts, empirical assignments have been made for the vibrations observed between 700 and 1700 cm^-1, A modified Urey-Bradley force field has been refined to satisfactorily reproduce the vibrational frequencies and isotopic shifts. of particular importance is the assignment of the normal modes in the structurally sensitive 1100–1300 cm^-1“fingerprint region” to specific combinations of C-C stretching and CCH rocking motions. The methyl-substituted^WC₈-C₉” and “C₁₂-C₁₃” stretches are highest in frequency at 1214 and 1248–1255 cm^-H, respectively, as a result of coupling with their associated C-methyl stretches. The C₈-C₉and C₁₂-C₁₃stretches also couple strongly with the C₁₀H and C₁₄H rocks, respectively. The 1169-cm^-1mode is assigned as a relatively localized C₁₀-C₁₁stretch, and the 1201-cm^-1mode is a localized C₁₄-C₁₅stretch. The frequency ordering and spacing of the C-C stretches in BR₅₆₈is the same as that observed in the all-trans-retinal protonated Schiff base. However, each vibration is -10 cm^-1higher in the pigment as a result of increased π-electron delocalization. The frequencies and Raman intensities of the normal modes are compared with the predictions of theoretical models for the ground- and excited-state structure of the retinal chromophore in bacteriorhodopsin.