Resonance Raman Studies of the HOOP Modes in Octopus Bathorhodopsin with Deuterium-Labeled Retinal Chromophores

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm⁻¹ for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm⁻¹ to C₁₀H and C₁₄H HOOP modes, and the band at 940 cm⁻¹ to C₁₁H=C_ı2H A_u-like HOOP mode. Except for a 26 cm⁻¹ downward shift, the C₁₁H=C₁₂H A_u-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C₁₀H and C₁₄H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C₇H and C_gH HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C₁₁H=C₁₂H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C₁₁=C₁₂ double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C₁₁H=C₁₂H A_u-like HOOP mode. The twisted nature of the chromophore, semi-quantitatively discussed here, likely affects the λ_max of the chromophore and its enthalpy. The nature of the HOOP modes of octopus bathorhodopsin differs substantially from those found in bovine bathorhodopsin. Thus, while the λ_max values for the two batho products are essentially the same as is their enthalpies (relative to their respective rhodopsins),. it seems likely that the various molecular factors that determine these quantities are present to different degrees. For example, the twist about C₁₁=C₁₂ double bond is likely larger in bovine bathorhodopsin than in octopus bathorhodopsin to account for the decoupling of C₁₂H wag from C₁₁H wag in bovine bathorhodopsin. This difference can be caused by the perturbation of a negatively charged protein residue in bovine bathorhodopsin (Eyring et al., 1982), which is much weaker in octopus bathorhodopsin. These issues are discussed in some detail.