PURIFICATION AND PROPERTIES OF REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE DEHYDROGENASE FROM PHOTOBACTERIUM-PHOSPHOREUM

Reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (EC 1.6.99.3) of Photobacterium phosphoreum was solubilized from membrane vesicles with 2% sucrose monolaurate, and was purified almost to homogeneity by use of diethylaminoethyl (DEAE)-Sephacel, hydroxylapatite, and Butyl-Toyopearl column chromatography in the presence of 0.5% sucrose monolaurate. About 100-fold purification was achieved, and the purified enzyme is composed of a single polypeptide with a molecular weight of 49000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The NADH dehydrogenase contains about one mol flavin adenine dinucleotide (FAD) as a prosthetic group per mol enzyme, but does not contain acid labile sulfide or non-heme iron. This NADH dehydrogenase can use ubiquinone-1, menadione, potassium ferricyanide, and cytochrome c as electron acceptors. The enzyme barely catalyzes the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. The enzyme is not susceptive to rotenone, 2-n-heptyl-4-hydroxyquinoline N-oxide, capsaicin, or N,N'-dicyclohexylcarbodiimide, which were reported as inhibitors of NADH dh I. On the other hand, flavone, reported as an inhibitor of NADH dh II of yeast and plant mitochondria, does inhibit the NADH dehydrogenase activity. These results indicate that the NADH dehydrogenase of P. phosphoreum belongs to NADH dh II. The NADH oxidase activity of the membrane vesicles of P. phosphoreum is remarkably stimulated by monovalent cations such as Na+ and K+, but is inhibited by divalent or trivalent cations. The NADH dehydrogenase activities (especially NADH-ferricyanide and NADH-ubiquinone-1 oxidoreductase) of the purified enzyme and membrane were also enhanced by Na+ and K+. However, the ubiquinol-1 oxidase activity of terminal oxidase complex (cytochrome bd complex) was not enhanced largely by the monovalent cations. These results suggest that in the respiratory chain of P. phosphoreum, NADH dehydrogenase contributes to the enhancement of electron transfer activity by monovalent cations.