Multicopper oxidases (MCO) couple the oxidation of a variety of substrates to the reduction of dioxygen to water (O₂ + 4e^- + 4H⁺ → 2H₂O). Electrons are transferred through a T1 copper near the substrate-binding site, to the trinuclear copper center (formed by two T3 coppers and a single T2 copper) where oxygen binds and is reduced. This mechanism of dioxygen reduction is not well understood and the key structures of the two reaction intermediates formed, called peroxide and native, remain elusive.

We have expressed and solved the crystal structure of CueO, a MCO found in E. coli. We have steady state kinetic measurements establishing Cu(I) as an excellent substrate for CueO and determined its binding site. Ability to convert Cu(I) to its less toxic form, Cu(II), could be one of the mechanisms by which CueO protects E. coli against copper toxicity.

We are devising methods for trapping all key intermediates in the CueO reaction cycle. To accomplish this, we have made mutations to key residues involved with catalysis to show transition between reaction intermediates. These include mutations like C500S and E506D. A collaborative project is underway to design a micro-flow cell device that will allow us to initiate controlled catalysis in the crystal, collect UV-Visible crystal absorption spectra and diffraction data for solving structures, all on the same crystal, greatly enhancing our ability to trap intermediates and correlate structure with spectra.