FOURIER TRANSFORM INFRARED SPECTRAL STUDIES ON THE SCHIFF BASE MODE OF ALL‐trans BACTERIORHODOPSIN and ITS PHOTOINTERMEDIATES, K and L*

Difference Fourier transform infrared spectra were recorded for bacteriorhodopsin upon irradiation at 230, 170 or 77 K, which gave, respectively, the spectrum of the M, L or K intermediate minus unphotolyzed all‐ trans bacteriorhodopsin (denoted as BR). By replacement of the Schiff base nitrogen with 15 N, or of either its hydrogen at N or C 15 with deuterium, the vibrational bands related to the Schiff base were identified and the isotope‐shifts evaluated for BR, K and L. The 1348 cm −l band of BR and K and the 1400 cm −1 band of L were sensitive to each of these isotope substitutions. The 1254 cm −1 band of BR, the 1245 cm −1 band of K and the 1301 cm −1 band of L were sensitive to either N‐ or C 15 ‐deuteration but not to 15 N‐substitution. The N—D in‐plane bending vibration of K and L appeared at 969 and 997 cm −1 , respectively, upon substitution with D 2 O. All the results show that L is larger in frequencies related to the N—H in‐plane bending vibration than K or BR and suggest that L has the strongest interaction with the protein. Among the bands containing an N—H bending vibration, the 1348 cm −1 band of K was more intense than the corresponding band of L at 1400 cm −1 . The C 15 ‐deuteration‐induced upshift of the 1245 cm −1 band of K was unobservable for the 1301 cm −1 band of L. Such differences between L and K might be brought about by a distortion in the retinal moiety close to the protonated Schiff base of the 13‐ cis chromophore.