Biochemical aspects of the visual process: XXXIX. Sulfhydryl group reactivity as a probe of transient protein conformational changes during rhodopsin photolysis

Abstract

1. 1.|The number of sulfhydryl groups available to 5,5′-dithiobis-(2-nitrobenzoic acid) during the photolysis of bovine rhodopsin in photoreceptor membranes has been determined. Under most conditions two sulfhydryl groups react per mole of rhodopsin, in agreement with earlier observations. However, an additional sulfhydryl group is detected if during illumination the chromophore is reductively linked to the protein with NaBH 4. This group does not arise from the cleavage of a disulfide bridge. 2. 2.|This phenomenon is transient and reduction with NaBH 4 at increasing time intervals after illumination results in a gradual disappearance of the additional sulfhydryl group. 3. 3.|In parallel, the reaction rate of the rhodopsin sulfhydryl groups towards 5,5′-dithiobis-(2-nitrobenzoic acid) is greatly increased in this reductively stabilized photointermediate. 4. 4.|It is suggested that this stabilized photointermediate represents a transitional state between metarhodopsin I and metarhodopsin II and then our observations indicate that sulfhydryl group reactivity transiently increases during the photolysis of rhodopsin. 5. 5.|In conclusion, the availability of an additional sulfhydryl group to 5,5′-dithiobis-(2-nitrobenzoic acid) is though to reflect transient protein conformational changes and/or changes in protein-lipid interactions during the photolysis of rhodopsin. Their maximal expression during the metarhodopsin I to metarhodopsin II transition suggests that these changes may play an essential role in the process of visual excitation.