Infrared studies of rhodopsin and its low temperature photoproducts, bathorhodopsin and isorhodopsin

Abstract

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes of the chromophore and protein that change in position and intensity between rhodopsin and the photoproducts formed at low temperature (70K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in identification of the vibrational modes, experiments were performed on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13c at carbon-15 or 15-deuterioretinal, or hydrated films of rod outer segments regenerated with retinal containing 13c at either carbon-10, carbon-11, carbon-13, or carbons-14 and -15. These infrared studies provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is all-trans-like. The positions of the C=N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis 15-deuterioretinal or 11-cis retinal containing 13c at carbon-15 are indicative that the Schiff base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C=N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C=N trans conformation in all three species, and that the ClO-Cll single bond is s-trans in bathorhodopsin. Finally, evidence is presented that, even in these early stages of the rhodopsin bleaching sequence, changes are occurring in the-opsin.