Fourier transform Raman study of retinal isomeric composition and equilibration in halorhodopsin

Earlier results suggested that the description of the photoreaction of halorhodopsin depends on our being able to distinguish and dissect the photoproducts of the all-trans chromophore from those of the 13-cis,15-syn chromophore. We have used FT Raman spectroscopy, a nonperturbing method, to analyze the isomeric states of the retinal under various conditions in halorhodopsins from Halobacterium salinarium and Natronobacterium pharaonis. The results indicate that light adaptation occurs in the former protein, and the amounts of the two isomers in the light-adapted and dark-adapted forms are consistent with the weights given to them, from spectroscopic and mathematical criteria, in the earlier calculations. No light adaptation occurs in the latter protein, which contains mostly, but not entirely, all-trans-retinal. There is no light adaptation in either protein in the absence of chloride, the transported substrate of these anion pumps, and in spite of a 13-cis-like photocycle, they contain both all-trans- and 13-cis, 15-syn-retinal under these conditions. These findings emphasize the need to consider the photoreactions of both isomers when studying halorhodopsin and provide a reliable method for determining the isomeric mixtures in this protein.