My work focuses on the multicopper oxidase (MCO) family of enzymes. This family includes enzymes found throughout biology including S. cerivisiae Fet3p and R. vernicifera laccase. All enzymes in this family catalyze the four electron reduction of dioxygen to water, which is coupled to the oxidation of substrate, which is specific to each particular MCO. In order to catalyze this reaction all MCO’s have a minimum of 4 coppers. These include a type 1 (blue) Cu site, which is the site of substrate oxidation and electron transfer to the trinuclear Cu cluster (made up of a type 2 Cu and coupled binuclear type 3 Cu site) located 13Ǻ away which serves as the site of dioxygen binding and reduction.

The primary aim of my research has been to use site directed mutagenesis to perturb the trinuclear cluster in Fet3p. Generating these mutated trinuclear clusters will allow us to study the contribution of the surrounding amino acids to the unique unsaturated coordination environment of the resting trinuclear cluster using a variety of spectroscopic techniques, including electronic absorption, electron paramagnetic resonance (EPR), circular dichroism (CD), and magnetic circular dichrosim (MCD). In addition to the resting state of the enzyme, we will use these mutations to gain more information about the reaction mechanism of the MCOs. Techniques such as stopped-flow absorption spectrophotometry will be used to study the reaction kinetics and rapid freeze-quench will be used to trap intermediates in the reaction coordinate which can then be further studied using EPR and MCD.