Fourier-transform infrared spectroscopy applied to rhodopsin. The problem of the protonation state of the retinylidene Schiff base re-investigated.

By measuring the rhodopsin--bathorhodopsin, isorhodopsin--bathorhodopsin, rhodopsin--isorhodopsin and rhodopsin--meta-II difference spectra with the method of Fourier-transform infrared spectroscopy we have identified the C = N stretching vibration of the protonated retinylidene Schiff base of rhodopsin, isorhodopsin and bathorhodopsin. In contrast to resonance Raman spectroscopy additional strong bands were observed between 1700 cm-1 and 1620 cm-1. Most of them depend on the isomeric state of the chromophore. The origin of these bands will be discussed. In the fingerprint region isorhodopsin and bathorhodopsin are quite similar but no similarities with infrared spectra of model compounds of any isomeric composition are observed. Therefore, no conclusions on the isomeric state of the retinal in bathorhodopsin can be drawn. We provide evidence for the modification of one or two carboxylic group(s) during the rhodopsin--bathorhodopsin and isorhodopsin--bathorhodopsin transition.