NMR Study of the Exchange Coupling in the Trinuclear Cluster of the Multicopper Oxidase Fet3p

Abstract

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) which oxidizes Fe2+to Fe3+. The electronic structure of the different copper centers in this family of enzymes has beenextensively studied and discussed for years with a particular focus on the exchange coupling regimein the trinuclear cluster (TNC). Using NMR spectroscopy we have quantified the exchange coupling constant in the type 3 center in a fully metalated oxidase; this value in Fet3p is significantly higher than that reported for proteins containing isolated type 3 centers as tyrosinase. We also provide evidence of exchange coupling between the type 2 and the type 3 Cu2+ ions, which supports the crystallographic evidence of dioxygen binding to the TNC. This work provides the foundation for the application of NMR to these complex systems.