Are C14-C15 single bond isomerizations of the retinal chromophore involved in the proton-pumping mechanism of bacteriorhodopsin?

Abstract

Resonance Raman spectroscopy is used to examine the possibility that C14 - C15 single bond isomerizations of the retinal prosthetic group are involved in the photochemical reactions of bacteriorhodopsin. Normal mode calculations show that the vibration that contains predominantly C14 - C15 stretch character is .apprxeq. 70 cm-1 lower in frequency in the 14-s-cis conformer than in the s-trans case. This geometric effect is insensitive to out-of-plane twists and should be observed in the sterically hindered 13-cis, 14-s-cis retinal protonated Schiff base, which has been proposed as the chromophore in the K and L intermediates of bacteriorhodopsin. Resonance Raman spectra were obtained of K625 by using the low temperature (77 K) spinning-cell technique. Isotopic substitutions with 13C and 2H show that significant C14 - C15 stretch character is observed in normal modes at .apprxeq. 1185-1195 cm-1. The relatively high frequency of the C14 - C15 stretch argues that K625 contains a 13-cis, 14-s-trans chromophore. Similarly, isotopic derivatives show that L550 has a localized C14 - C15 stretch at 1172 cm-1, consistent with a 14-s-trans chromophore. These results argue that the primary step in bacteriorhodopsin is a C13 .dbd. C14 trans .fwdarw. cis photoisomerization that does not involve C14 - C15 s-cis structures.