STOICHIOMETRY AND SPECTROSCOPIC IDENTITY OF COPPER CENTERS IN PHENOXAZINONE SYNTHASE - A NEW ADDITION TO THE BLUE COPPER OXIDASE FAMILY

Phenoxazinone synthase catalyzes the oxidative condensation of two molecules of substituted o-aminophenols to the phenoxazinone chromophore of actinomycin. Cyclization occurs with the concomitant reduction of molecular oxygen to water. We have shown that the enzyme requires 4-5 copper atoms/monomer for full activity and the additional copper inhibits the enzyme. The optical absorption spectrum of phenoxazinone synthase is also dependent on the Cu per monomer ratio, and the absorption peak at 598 nm has a maximum extinction coefficient of 4000 +/- 150 M-1 cm-1 at a ratio of 4-5 Cu atoms per monomer. The electron paramagnetic resonance (EPR) spectrum of enzyme as isolated with low copper content (0.8 Cu/monomer) only shows the presence of type 1 (blue) copper centers (g(parallel-to) = 2.24, A = 0.0067 cm-1, and g(perpendicular-to) = 2.07). Enzyme incubated with 4-5 Cu per monomer demonstrates the presence of both type 1 and type 2 copper centers with a stoichiometry of one type 1 center per monomer and the remainder bound as type 2 Cu2+. Anaerobic incubation of substrate with enzyme containing five Cu atoms per subunit results in bleaching of the blue center. The EPR spectrum of the enzyme reduced under these conditions suggests that one of the type 2 Cu2+ centers with a g(parallel-to) = 2.34, A = 0.016 cm-1, and g(perpendicular-to) = 2.07 remains oxidized and is not involved in catalysis. From the spectroscopic data in this paper, phenoxazinone synthase appears to contain three functional copper atoms that can accept electrons from substrate and two additional copper atoms whose function has yet to be defined. The copper content and spectroscopic behavior of phenoxazinone synthase appear similar to many enzymes of the blue copper oxidase family