Spectroscopic studies of multicopper oxidases: Probing the nature and reactivity of the different copper sites

Multicopper oxidases are a class of enzymes that couple four one-electron substrate oxidations with the four-electron reduction of dioxygen to water. This reaction is accomplished using a copper active site comprised of type one, type two, and type three copper sites. A variety of spectroscopic methods, including electronic absorption, circular dichroism, magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopy, are used to characterize the geometric and electronic structure of the different copper sites in multicopper oxidases. The electronic structure is then related to the reactivity properties of each site (e.g. its involvement in electron transfer vs. dioxygen reduction). The type one copper site in fungal laccases is found to have a unique trigonal planar geometry, a strong copper-cysteine bond, a high redox potential, and a strong ligand field. The active site environment was probed using site directed mutagenesis and the axial ligand was found to play an important role in tuning the properties and reactivity of the type one copper site. In multicopper oxidases the type two and type three copper sites combine to form a trinuclear copper cluster that is responsible for activating dioxygen. The trinuclear copper cluster was studied in two multicopper oxidases in which the type one copper was removed. Removal of the type one copper facilitated spectroscopic analysis of the trinuclear cluster and enabled a peroxide intermediate produced upon reaction of the reduced enzyme with dioxygen, to be trapped. Spectroscopic and kinetic studies on this peroxide intermediate revealed that it decays very slowly because there is a large Franck-Condon barrier to reductively cleave the peroxide O-O bond by one electron. In the native enzyme, where all four copper ions are present, this peroxide intermediate is predicted to decay via a two electron reductive cleavage of the O-O bond. This process is predicted to be 107 times faster because the increased driving force for the two-electron process lowers the enthalpic contribution to the Franck-Condon barrier. These results support a molecular mechanism in the multicopper oxidases in which the reduction of dioxygen to water proceeds by two two-electron steps.