Tunable laser resonance raman spectroscopy of bacteriorhodopsin.

Bacteriorhodopsin is a rhodopsin-like protein found in the cell membrane of Halobacterium halobium. It shows an absorption maximum at 570 nm and, in the light, undergoes cyclic spectral changes which include a relatively long-lived complex absorbing maximally at 412 nm. Excitation profiles have been obtained with several laser frequencies for two vibrations in the resonance Raman spectrum of bacteriorhodopsin. The results show that the Schiff base retinylidene lysine linkage is protonated in the 570 nm complex and that in the 412 nm complex it is unprotonated. The 412 nm complex must be present at appreciable concentrations when bacteriorhodopsin is exposed to high-energy argon ion laser light of the Raman spectrophotometer at room temperature. We conclude that the observed C=N stretch at 1622 cm(-1) in the room temperature spectra, which in an earlier study by Mendelsohn was interpreted as evidence for an unprotonated linkage in bacteriorhodopsin, results from the presence of the 412 nm complex.