Spectroscopic and Kinetic Studies on the Oxygen-centered Radical Formed during the Four-electron Reduction Process of Dioxygen by Rhus vernicigera Laccase

The oxygen-centered radicalbound to the trinuclear coer center was detected as intermediate during the reoxidation process of the reduced Rhus vernicifera laccase with dioxygen and characterized by using absorption ,stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies and by super conducting quantum interface devices measurement. The intermediate bands appeared at 370 nm (e～1000), 420nm (sh), and 670 nm (weak) within 15ms, and were observable for～2 min at pH7.4 but for less than 5s at pH 4.2. The first-order rate constant for the decay of the intermediate has been determined by stopped-flow spectroscopy, showing the isotope effect, kH/kD of 1.4 in D2O. The intermediate was found to decay mainly from the protomated from by analyzing pH dependences. The enthalpy and entropy of activation suggested that a considerable structure change takes place around the active site during he decay of the intermadiate. The EPR spectra at cryogenic temperatures (<27) showed wo broad signals with g～1.8 and 1.6 depending on pH. We propose an oxygen-centered radical in magnetic interaction with the oxidized type ３copper ions as the structure of the three-electron reduced form of dioxygen.