Bacteriorhodopsin's L550 intermediate contains a C14-C15 s-trans-retinal chromophore

Conformational changes of the retinal chromophore about the C14-C15 bond in bacteriorhodopsin (BR) have been proposed in models for the mechanism of light-driven proton transport. To determine the C14-C15 conformation in BR's L550 intermediate, the authors have examined the resonance Raman spectra of BR derivatives regenerated with retinal deuterated at the 14 and 15 positions. Vibrational calculations show that the C14-2H and C15-2H rocking modes form symmetric (A) and antisymmetric (B) combinations in [14,15-2H]retinal chromophores. When there is a trans conformation about the single bond between C14 and C15 (14-s-trans), a small frequency separation or splitting is observed between the A and B modes. In addition, the monodeuterium rock should appear at an unusually low frequency in the 14-2H-labeled 14-s-cis molecules. These patterns are insensitive to computational details. Time-resolved resonance Raman spectra were obtained of BR's L550 intermediate regenerated with [14-2H]-, [15-2H]- and [14,15-2H]retinal. These results show that L550 contains a 14-s-trans chromophore and suggest that only 14-2-trans structures are involved in the proton pumping photocycle of BR