Spectroscopic and Kinetic Studies on the Oxygen-centered Radical Formed during the Four-electron Reduction Process of Dioxygen byRhus vernicifera Laccase

The oxygen-centered radical bound to the trinuclear copper center was detected as an 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 (epsilon approximately 1000), 420 nm (sh), and 670 nm (weak) within 15 ms, and were observable for approximately 2 min at pH 7.4 but for less than 5 s 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, k(H)/k(D) of 1.4 in D(2)O. The intermediate was found to decay mainly from the protonated form by analyzing pH dependences. The enthalpy and entropy of activation suggested that a considerable structure change takes place around the active site during the decay of the intermediate. The EPR spectra at cryogenic temperatures (<27 K) showed two broad signals with g approximately 1.8 and 1.6 depending on pH. We propose an oxygen-centered radical in magnetic interaction with the oxidized type III copper ions as the structure of the three-electron reduced form of dioxygen.