Resonance Raman studies on the intermediate K-590 in the photocycle of bacteriorhodopsin

Resonance Raman (RR) spectra of the intermediate K-590 in the photocycle of the retinylidene Schiff base chromophore of bacteriorhodopsin (BR) were recorded at 21{degree}C, where K has a lifetime of {approximately}2 {mu}s. An aqueous suspension of purple membranes from Halobacteria (16 {mu}M concentration of BR) was flowed across the waist of a laser beam (514 nm) serving for both photolysis and RR excitation. The residence time of the sample in the beam waist was {approximately}2 {mu}s. To subtract the contribution of the parent BR-570, a special experiment was designed in which two spectra, one at a low degree of photolysis (mainly consisting of BR-570) and one at a significantly higher degree of photolysis (mixture of BR-570 and K-590) could be recorded simultaneously. This allowed us to perform the difference procedure with high accuracy. New vibrational features in the spectrum of K-590 were identified in the C{double bond}C stretching region. From a comparison with the RR spectra of the parent BR-570 and the subsequent product L-550 the following was concluded: During the photoinduced transition from BR-570 to K-590 a negatively charged counterion is removed from the positively charged Schiff base group.