Flavin Mononucleotide: The Coenzyme of Reduced Diphosphopyridine Nucleotide Dehydrogenase

From the particulate reduced nicotinamide adenine dinucleotide (DPNH) oxidase of beef heart, Mackler (3) has obtained a soluble DPNH dehydrogenase which was estimated to be approximately 90 to 95% homogenous when examined electrophoretically.l Similarly, when the enzyme was chromatographed on diethylaminoethyl cellulose with phosphate buffer, pH 7.5, in a gradient elution method (0.001 M to 0.01 M), only a single, symmetrical peak with respect to protein and flavin was observed in the elution profile (4). The enzyme contains bound flavin and nonheme iron in the ratio of 1: 2. The minimal molecular weight, calculated from the flavin content, is 100,000 which is in general agreement with a value of 80,000 to 90,000 obtained by sedimentation in the ultracentrifuge.’ The activity of the enzyme with 2,6-dichlorophenolindophenol as an electron acceptor is 40 pmoles of DPNH oxidized per minute per mg of protein. With ferricyanide and cytochrome c as acceptors, the DPNH oxidizing activities are 40 and 3, respectively, in the same units. Although this enzyme is similar to other DPNH dehydrogenases described previously (5-ll), it is difficult to compare the properties (molecular weight, pH optimum, turnover number, specificity for electron acceptors, etc.) of these various enzymes since, even when prepared from the same tissue, different methods of solubilization have been used. One of the principal discrepancies between the various DPNH dehydrogenases is the nature of the flavin component. It has been reported that only FAD is found in a soluble DPNH dehydrogenase obtained by treatment of beef heart electron transport particle with Naja nuja venom (7, 9), and FAD has also been identified in a lipoflavoprotein DPNH dehydrogenase from beef heart (12). Earlier, Mahler et al. (5) had reported that the prosthetic group of the soluble DPNH-cytochrome c reductase from pig heart is a flavin dinucleotide not identical with FAD. On the other hand, we have reported in preliminary communications (1, 2) that FMN2 is the flavin component of the soluble DPNH dehydrogenase prepared from DPNH osidase, and King3 has reached