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 mol ecules of substituted o-aminophenols to the phenoxazinone chromophore of actinomycin. Cyclization occurs with the concomitant reduction of m olecular oxygen to water. We have shown that the enzyme requires 4-5 c opper atoms/monomer for full activity and the additional copper inhibi ts the enzyme. The optical absorption spectrum of phenoxazinone syntha se is also dependent on the Cu per monomer ratio, and the absorption p eak 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 paramagneti c resonance (EPR) spectrum of enzyme as isolated with low copper conte nt (0.8 Cu/monomer) only shows the presence of type 1 (blue) copper ce nters (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 pr esence of both type 1 and type 2 copper centers with a stoichiometry o f one type 1 center per monomer and the remainder bound as type 2 Cu2. Anaerobic incubation of substrate with enzyme containing five Cu ato ms per subunit results in bleaching of the blue center. The EPR spectr um of the enzyme reduced under these conditions suggests that one of t he type 2 Cu2+ centers with a g(parallel-to) = 2.34, A = 0.016 cm-1, a nd g(perpendicular-to) = 2.07 remains oxidized and is not involved in catalysis. From the spectroscopic data in this paper, phenoxazinone sy nthase appears to contain three functional copper atoms that can accep t electrons from substrate and two additional copper atoms whose funct ion has yet to be defined. The copper content and spectroscopic behavi or of phenoxazinone synthase appear similar to many enzymes of the blu e copper oxidase family