EFFECT OF CHLORIDE ON THE THERMAL REVERSE REACTION OF INTERMEDIATES OF IODOPSIN

Among the intermediates in the bleaching process of iodopsin, a chicke n red-sensitive cone visual pigment, the bathe and meta I intermediate s (bathe and meta I) formed at low temperatures revert to the original iodopsin by thermal reactions [Yoshizawa & Wald (1967) Nature 214, 56 6-572; Imamoto, Imai, Yoshizawa, & Shichida (1994) FEES Lett, 354, 165 -168]. In order to elucidate the relationship between Cl- binding to i odopsin and these reverse reactions, we have prepared a sample of iodo psin whose Cl--binding site is vacant (anion-unbound iodopsin) and com pared the thermal reactions of its bathe and meta I intermediates with those of Cl--bound (native) and nitrate-bound iodopsins. The reverse reaction from bathe is observed in both Cl--bound and anion-unbound io dopsins, while the reaction from meta I is observed only in Cl--bound iodopsin. These results indicate that Cl- binding is indispensable for the reverse reaction from meta I, but not from bathe. The reverse rea ction from meta I has been further investigated as a function of Cl- c oncentration, and the dissociation constant of Cl- in meta I is estima ted to be similar to 20 mM. This value is about 200 times larger than that of iodopsin (0.1 mM), and close to the physiological Cl- concentr ation in photoreceptor cells, suggesting that Cl- could be released fr om the protein moiety during the bleaching of iodopsin.