CHEMOENZYMATIC SYNTHESIS OF 11-CIS-RETINAL PHOTOAFFINITY ANALOG BY USE OF SQUID RETINOCHROME

We expect the photoactive 11cis-3-diazo-4-oxoretinal ( 1) to be crucial in clarifying the visual transduction process, but its synthesis has not been successful. However, as described below, the preparation has been achieved by incubation of trans isomer 2with squid retinochrome which smoothly performed the critical transf 11-cis isomerization to yield1. The visual transduction process is initiated by isomerization of the 11-cis-retinal chromophore in rhodopsin to all-trans, cleavage of the chromophore/opsin bond and terminates in alltrans-retinal and opsin. 1 Thecisf trans isomerization triggers a chain of conformational changes in the opsin which induces an enzymatic cascade leading to vision. 1a,2 Scheme 1 depicts the intermediates which have been identified based on lowtemperature spectroscopy. 3 However, the movement of the chromophore relative to the receptor opsin along the isomerization pathway remains unknown. Our objective is to clarify this crucial aspect on a molecular structural basis using photoaffinity labeling as the main tool. Photolysis of a pigment incorporating the nonisomerizable 11cis-locked retinal analog3 (Scheme 1) resulted in clear-cut cross-linking to Leu-266, thus revealing that the ionone C-3 is in close contact with helix F of rhodopsin in the dark. 4 On the other hand, studies using a photoreactive 11cis-retinal analog in which the 11-ene is not locked showed that the C-3 region cross-linked to both helices C and F; 5 recent studies with spin labels6 also showed that movements of these two helices were involved in the light activation process. It is thus possible that thecisf trans isomerization results in a “flip-over motion of the ring” from the proximity of helix F to helix C as well as movements of helices F and C and that this induces the conformational changes responsible for the enzymatic cascade. This scheme has been further corroborated by Sakmar and coworkers who have demonstrated that the relative movements of helices C and F is required for activation of the G-proteincoupled receptor rhodopsin. 7