A bacterial quercetin 2,3-dioxygenase has been recently characterized. This enzyme catalyzes the oxidative cleavage of the substrate quercetin. It belongs to the cupin superfamily of proteins defined by a beta barrel structure produced by a conserved cupin sequence. This motif provides metal ion ligands (3 His and 1 Glu). Quercetin 2,3-dioxygenase has been found to be active with a number of divalent metal ions including: Fe2+, Mn2+, Co2+, Cu2+, and Ni2+, but Mn2+ may be the preferred cofactor as shown by occupancy and activity evidence. Metal substituted proteins have been generated previously by growing the organism in minimal media supplemented with a specific metal. However, this protocol does not provide complete metal incorporation. A new protocol for the generation of metal substituted enzyme based on unfolding the protein then refolding it in the presence of metal ions has proven to generate active enzyme. The results of this new protocol will be presented.