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

The rate of regeneration of rhodopsin, from I I-cis-retinal and opsin, and bacteriorhodopsin from all-transretinal and bacterio-opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo-protein via a Schiffs base linkage since after treatment with NaBH4 an active apo-protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9 4 or 11-cis retinal up to carbon atom 11. These C13 and CIS molecules were not distinguished between aldehyde, ketone or alcohol functional groups.The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all-trans C1 d d e h y d e (j-ionylideneacetaldehyde) or c 1 8 -ketone (/I-ionylidenepent-3-ene-2-one) which, compared to retinal, lack two or three carbon atoms from the end of the polyene chain. The inhibition was very dependent upon the presence of the all-trans isomer and required aldehyde or ketone as functional group; nitriles and alcohols were less effective. However, similarly to retinol, the all-trans CI7 and c 1 8 alcohols underwent a bathochromic shift and showed fine-structured spectra when mixed with bacterio-opsin

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