Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Abstract

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor inHalobacterium halobium.We have obtained resonance Raman vibrational spectra of the native membranebound form of SRss7 and used these data to determine the structure of its retinal prosthetic group.The similar frequencies and intensities of the skeletal fingerprint modes in SRSs7, bacteriorhodopsin (BRses), and halorhodopsin (HR678) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm") demonstrate that the retinal chromophore has an all-trans configuration.The shift of the C=N stretching mode from 1628 cm" in HzO to 1620 cm" in DzO demonstrates that the chromophore in SRsa7 is bound to the protein by a protonated Schiff base linkage.The small shift of the 1195 cm" Cl4-C16 stretching mode in D20 establishes that the protonated Schiff base bond has an anti configuration.The low value of the Schiff base stretching frequency together with its small 8 cm" shift in D20 indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion.This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.Sensory rhodopsin I (SR-I),' halorhodopsin (HR), and bacteriorhodopsin (BR) are retinal proteins found in the cytoplasmic membrane of the bacterium Halobacterium halobium (Lanyi, 1986;Spudich and Bogomolni, 1988;Stoeckenius and