Developing the mechanism of dioxygen reduction catalyzed by multicopper oxidases using protein film electrochemistry

We present electrochemical and computational evidence of additional features in the mechanism for the reduction of O-2 catalyzed by enzymes known as the multicopper oxidases (MCOs). Using protein film electrochemistry, we show that the dormant Resting Oxidized state of the enzyme must be in equilibrium with a catalytically active form. In four different MCOs turning over O-2 at pH values several units above their respective optima, we observed electrocatalytic behavior consistent with a second reversible resting state that lies outside the main catalytic cycle (the X State). We describe how electrochemistry alone can be used to determine all the rate constants incorporated in our expanded mechanism for MCO-catalyzed O-2 reduction, using fully catalytically competent enzyme. We compare high-potential MCOs (laccases from Trametes versicolor and Coriolopsis gallica, and bilirubin oxidase from Myrothecium verrucaria) and low-potential MCOs (laccase from Rhus vernicifera and CotA from Bacillus subtilis), and explain how the same scheme and analysis can be used in both cases, even though specific rate constants vary widely between species. For example, the rate of formation of the Resting Oxidized state is similar to 1000 times faster in high-potential MCOs than in low-potential analogues. In contrast, all the MCOs showed similar X State fractions. For laccase from Trametes versicolor, about a quarter of the enzyme molecules occupy the X State during catalysis at high pH values. These findings are elucidated through computational finite time difference modeling and numerical analysis, which allows the X State to be positioned in the catalytic cycle as a dead-end state from the Peroxy Intermediate.