Assignment and interpretation of hydrogen out-of-plane vibrations in the resonance Raman spectra of rhodopsin and bathorhodopsin.

Resonance Raman studies of visual pigment analogues regenerated with 10-D, 10,l 1-D2, 11-D, 1 1,12-D2, 12-D, 15-D, and 20,14-D4 isotopic derivatives of retinal have been used to assign the intense hydrogen outsf-plane (HOOP) vibrations in the spectra of rhodopsin, isorhodopsin, and bathorhodopsin. Low temperature 77 K photostationary steady-state experiments show that the unique 853-, 875-, and 921-cm-' lines of bathorhodopsin are due to isolated 14, 10, and 1 1 hydrogen out-of-plane wagging modes, respectively. The 12 HOOP wag could not be directly observed but must lie in the 830-860-cm-' region, an unusually low frequency for this vibration. Room temperature rapid-flow experiments similarly demonstrate that the 969-cm-' line in rhodopsin is due to the coupled Cl1H=CI2H A2 HOOP. The 959-cm-' line in isorhodopsin is attributed to a C7H=C8H A,, HOOP. For bathorhodopsin, the small shift of the 11 HOOP wag with 12-D substitution (921 916 cm-') clearly indicates that the 11 and 12 HOOPS are only weakly coupled. This contrasts I t has long been known that light absorption in the visual pigment rhodopsin results in the cis trans isomerization of the covalently bound 1 1-cis-retinal chromophore (I) (Wald,