Time-resolved resonance Raman characterization of the bL550 intermediate and the two dark-adapted bRDA/560 forms of bacteriorhodopsin.

The resonance Raman spectrum of the second intermediate in the bacteriorhodopsin cycle, bL550, is obtained by a simple flow technique. The Schiff base linkage in this intermediate appears to be protonated, contrary to previous suggestion. The fingerprint region of the spectrum of bL550 does not closely match those of any presently available model Schiff bases of retinal isomers, though some comparisons can be made. The resonance Raman spectrum of dark-adapted bacteriorhodopsin is obtained and decomposed by computer subtraction of the spectrum of bR570. The remaining spectrum does not match the spectra of any model compounds presently in the literature. The spectra of bL550 and dark-adapted bRDA/560 from purple membrane in H2O are compared to those in D2O. It is found that changes in the spectrum occur in the 1,600 - 1,650 cm-1 region as well as in the 800 - 1,000 cm-1 region, but apparently not in the fingerprint region (1,100 - 1,400 cm-1). The possibilities of conformational changes of the retinal chromophore in the light adaptation process as well as the photosynthetic cycle are discussed.