Complete assignment of the hydrogen out-of-plane wagging vibrations of bathorhodopsin: chromophore structure and energy storage in the primary photoproduct of vision.

Resonance Raman vibrational spectra of the retinal chromophore in bathorhodopsin have been obtained after regenerating bovine visual pigments with an extensive series of 13C- and deuterium-labeled retinals. A low-temperature spinning cell technique was used to produce high-quality bathorhodopsin spectra exhibiting resolved hydrogen out-of-plane wagging vibrations at 838, 850, 858, 875, and 921 cm-1. The isotopic shifts and a normal coordinate analysis permit the assignment of these lines to the HC7 = C8H Bg, C14H, C12H, C10H, and C11H hydrogen out-of-plane wagging modes, respectively. The coupling constant between the C11H and C12H wags as well as the C12H wag force constant are unusually low compared to those of retinal model compounds. This quantitatively confirms the lack of coupling between the C11H and C12H wags and the low C12H wag vibrational frequency noted earlier by Eyring et al. [(1982) Biochemistry 21, 384]. The force constants for the C10H and C14H wags are also significantly below the values observed in model compounds. We suggest that the perturbed hydrogen out-of-plane wagging and C-C stretching force constants for the C10-C11 = C12-C13 region of the chromophore in bathorhodopsin result from electrostatic interactions with a charged protein residue. This interaction may also contribute to the 33 kcal/mol energy storage in bathorhodopsin.