The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids