Mixed Function Oxidation: V. FLAVIN INTERACTION WITH A REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE DEHYDROGENASE, ONE OF THE ENZYMES PARTICIPATING IN CAMPHOR LACTONIZATION

Abstract

Abstract 1. The isolation of a pure reduced diphosphopyridine nucleotide:(acceptor) oxidoreductase from Pseudomonas putida, strain C1B, in 15% yield is described. 2. The absorption spectrum, fluorescence spectrum, and lack of activity with dye electron acceptors show the isolated enzyme to be devoid of flavin (apoenzyme). Iron, copper, and molybdenum are also absent. 3. One mole of the apoenzyme, molecular weight 36,000, will bind 1 mole of flavin mononucleotide, 2 moles of flavin adenine dinucleotide, or 1 of each when presented with them at equal concentrations. Two flavin-binding sites are present; one is common to both flavins and is catalytically active. The other is specific for flavin adenine and is not involved in catalysis. Equal concentrations of these two flavins are present in crude cell extracts; riboflavin is absent. 4. The dissociation constant, Kd, for the apoenzyme-flavin complex is 4.5 x 10-7 m. The minimum turnover number for reduction of the complex by reduced phosphopyridine nucleotide is 30,000. Michaelis constants, Km, for interaction of free flavin mononucleotide and reduced diphosphopyridine nucleotide with the complex are 3 x 10-6 m and 10-4 m, respectively. 5. The dissociation constants for the apoenzyme-2 flavin adenine dinucleotide complex lie within the range 2.5 to 7 x 10-6 m. A minimum turnover number for reduction of this complex is 39,000. Michaelis constants for interaction of free flavin adenine and reduced diphosphopyridine nucleotide with the complex are 1.9 x 10-5 m and 2.1 x 10-4 m, respectively. 6. Sulfhydryl groups and iron are not involved in flavin or reduced diphosphopyridine nucleotide binding; reduced diphosphopyridine nucleotide is not bound in the absence of flavin. Oxidation and reduction of an enzyme sulfhydryl group are not involved in catalysis. 7. Reduction of the apoenzyme-flavin complex proceeds by two 1-electron transfer steps. The first, more rapid transfer yields the flavin semiquinone. Reoxidation of the fully reduced complex proceeds by a single 2-electron transfer when free flavin mononucleotide is the electron acceptor. 8. A possible role for the flavin adenine bound to the specific site in coupling this enzyme to an iron-containing mixed function oxidase, which it is known to supply with electrons, is envisaged.