Determination of Retinal Schiff Base Configuration in Bacteriorhodopsin

Resonance Raman spectra of the BR568, BR548, K625, and L550intermediates of the bacteriorhodopsin photocycle have been obtained in1H2O and2H2O by using native purple membrane as well as purple membrane regenerated with 14, 15-13C2and 12,14-2H2isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C14--C15stretch to the normal modes in the 1100-to C14--C15fingerprint region and to characterize the coupling of the C14--C15stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C==N cis configuration the C14--C15stretch and the NH rock are strongly coupled, resulting in a large (≈ 50-cm-1) upshift of the C14--C15stretch upon deuteration of the Schiff base nitrogen. In the C==N trans geometry these vibrations are weakly coupled and only a slight (