Spectroscopic studies on the mechanism of the topa quinone generation in bacterial monoamine oxidase.

Electron paramagnetic resonance (EPR), circular dichroism (CD), and optical absorption spectroscopies have been used to investigate the copper-dependent autoxidation process generating the 6-hydroxydopa (topa) quinone cofactor in the recombinant phenethylamine oxidase from Arthrobacter globiformis. The cupric ion bound to the copper/topa quinone-less, inactive enzyme is first reduced to Cu(I), as inferred from the spectroscopic features observed under strictly anaerobic conditions. Cu(I) is also detectable chemically with a Cu(I)-specific chelating agent, bathocuproinedisulfonate. Introduction of a limited amount of oxygen then leads to the formation of a paramagnetic species (g = 2.004) that is stable for over several to 10 min but vanishes swiftly upon addition of sufficient oxygen. Strikingly, the hyperfine EPR structure of the organic radical is almost identical with that of the topa semiquinolamine observed in the copper/topa quinone-containing, active enzyme anaerobically reduced with substrate. Concomitant with the generation of topa quinone exhibiting characteristic optical absorption and CD bands under fully aerobic conditions, the bound copper finally shows EPR signals typical of nonblue type II Cu(II) and optical absorption around 700 nm with negative CD above 700 nm. None of these spectral changes are evoked in the binding of Cu(II) to the Tyr382-->Phe mutant enzyme, indicating that the precursor Tyr382 to topa quinone participates in the initial reduction of bound copper and serves as the origin of the transiently formed semiquinone radical. The prosthetic cupric ion plays an essential role, by changing its redox state, in the oxidative modification of the tyrosyl phenol ring, leading to topa quinone.