STRUCTURAL-CHANGES IN EARLY PHOTOLYSIS INTERMEDIATES OF RHODOPSIN FROM TIME-RESOLVED SPECTRAL MEASUREMENTS OF ARTIFICIAL PIGMENTS STERICALLY HINDERED ALONG THE CHROMOPHORE CHAIN

Kinetic spectra of early photolysis intermediates were monitored after nanosecond laser photolysis of a series of artificial visual pigments containing retinal analogs with bulky substituents along the polyene chain. Time-resolved absorbance changes over the spectral range 400-70 0 nm were recorded at discrete times from 20 ns to 5 mu s following ro om temperature excitation with a pulse of 477 nm light. Photolysis of bovine rhodopsin regenerated with 9-ethyl-9-cis-retinal, 19,11-ethano- 11-cis-retinal, or 13-ethyl-9-cis-retinal produced intermediates simil ar to those seen after rhodopsin photolysis, i.e. bathorhodopsin (Bath o) reversible arrow blue-shifted intermediate (BSI) --> lumirhodopsin (Lumi). In contrast to previously studied artificial pigments and rhod opsin itself, for these chromophores with bulky substituents, the equi librium between Bathe and BSI is back-shifted. The stability of BSI re lative to Bathe is most affected in the 13-ethyl pigment, which had an equilibrium constant of 0.4, approximately one-third of the value obs erved for rhodopsin. As the bulky substituent moves toward the C9 end of the chromophore, K-eq moves toward the rhodopsin value, with the re sult for the locked 9-trans-rhodopsin pigment being intermediate betwe en those of the 9-ethyl and 13-ethyl pigments. The presence of bulky s ubstituents also slows the microscopic rate of Bathe decay. This effec t is largest for the 9-ethyl pigment whose Bathe decay is slowed by a factor of 5. Freedom of movement of the 9-methyl (restricted in the 9- ethyl case) is proposed to control the rate of Bathe decay in a mechan ism involving passage of the chromophore's C8-hydrogen by the 5-methyl group of its beta-ionone ring to form BSI. For all these pigments, th e decay of BSI is substantially slower than for previous pigments, ind icating that steric hindrance along the polyene chain interferes with the protein change triggered by BSI formation and suggesting that the protein change may involve side chains adjacent to this region of the chromophore.