Specificity of the retinal binding site of bacteriorhodopsin: chemical and stereochemical requirements for the binding of retinol and retinal.

Abstract

The complexes formed from bacteriopsin and various retinyl compounds were analyzed by fluorescence and absorption spectroscopy. The binding of retinol occurs in two steps. In the first reaction the molecule is fixed in the retinal binding site of the protein. In this state, energy transfer from aromatic amino acid residues to the retinyl moiety is observed. all-trans-Retinal and the 13-, 11-, and 9-cis-retinols are bound in the chromophoric site. In the second reaction the cyclohexene ring and the side chain of the retinyl moiety are forced into a planar conformation. This reaction is mediated by a base (B1) with a pK of 3.8 and requires the oxygen atom but not the free hydroxyl group of retinol, indicating interaction with a group AH (pK greater than or equal to 10.5). The ring-chain planarization reaction is blocked for the 9-cis isomer of retinol. Binding studies with bacterioopsin and retinal isomers reveal that, as in the case of the corresponding retinols, B1 mediates ring-chain planarization in the case of the all-trans, 13-cis, and 11-cis isomers but not with the 9-cis isomer. Reconstitution of the purple complex from the intermediate 430-460-nm chromophore requires the presence of a second base (B2) with a pK of 4.6. This reaction is exclusive for all-trans- and 13-cis-retinal