Effect of Nicotinamide Adenine Dinucleotide on the Oxidation-Reduction Potentials of Lipoamide Dehydrogenase from Pig Heart

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Abstract
The effect of NAD + on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH 2 couple in the presence on NAD + were −218 mV and −251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD + binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD + Fluorescence quenching of bound FAD by NAD + was released by phenobarbital. The results suggest that NAD + strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD + acts not only as an acceptor of electrons from EH 2 , but also as an effector in the flavin-disuluide interaction of EH 2 .