Optical Activity and Conformation Studies of Pig Heart Lipoamide Dehydrogenase

Abstract Flavin adenine dinucleotide has been dissociated from pig heart lipoamide dehydrogenase in the presence of 1.5 m guanidine hydrochloride. Measurements of the circular dichroism on the apoenzyme show evidence of a large change in conformation, based on the molar ellipticity in the region 240 to 200 mµ. When FAD is removed from the holoenzyme in guanidine, but in the presence of approximately equimolar FMN, no change appears in the circular dichroism of the apoenzyme in the 200 mµ region. The ultraviolet absorption of the apoprotein indicates that no FMN remains bound to the apoenzyme. Either FMN stabilizes the protein in the presence of guanidine or, after the loss of FAD, promotes the return of a conformationally different species to the native form of the enzyme. Lipoamide dehydrogenase activity is reconstituted in the presence of FAD and dithiothreitol.