Resonance Raman Spectroscopy of an Ultraviolet-Sensitive Insect Rhodopsin

We present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11 -ds-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10 °C in both H₂0 and D₂0. The C=N stretching mode at 1660 in H₂0 shifts to 1631 upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100 °C with 406.7-nm excitation, to enhance scattering from rhodopsin 345 nm), contain a significant contribution from a small amount of contaminants [cytochrome(s) and/or accessory pigment] in the sample, the C=N stretch at 1664 suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at ~ 1660 in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.