Studies on Monooxygenases

Imidazole acetate monooxygenase, which catalyzes the hydroxylation of imidazole acetate, was purified about 250-fold from extracts of induced cells of a pseudomonad and was crystallized. The molecular weight was determined to be approximately 90,000, and the molecular activity was calculated to be 2,260 at 24°. The enzyme was shown to be homogeneous upon acrylamide gel electrophoresis and ultracentrifugation. The crystalline enzyme showed an absorption spectrum characteristic of a flavoprotein with absorption maxima at 270, 383, and 442 mµ. The flavin component of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of enzyme. Aerobic incubation of the enzyme with imidazole acetate and reduced nicotinamide adenine dinucleotide resulted in the accumulation of an unstable intermediate having an absorption spectrum with a maximum at 259 mµ, which was indistinguishable from that of imidazolone acetate. Flavin bound to the enzyme was reduced by the anaerobic addition of a stoichiometric amount of reduced nicotinamide adenine dinucleotide. The reduced form of enzyme-bound flavin was found to serve as an electron donor for the hydroxylation reaction. Some other properties of the enzyme are also described.