In an effort to characterize mitochondrial glyoxalase II from A. thaliana, GLX 2-5, was cloned and over-expressed in Escherichia coli. This is the first time a plant mitochondrial glyoxalase II has been heterologously over-expressed in E.coli. GLX 2-5 was overexpressed in rich media supplemented with iron, zinc, both iron and zinc, and no added metal. Although, metal analysis showed differences in metal ion composition among the enzymes produced under different conditions, no significant difference in catalytic activity was observed. EPR spectroscopy indicated the presence of two dinuclear metal centers; Fe(III)- Zn(II) and Fe(III)-Fe(II). ¹H NMR spectroscopy indicated the binding of Fe(II) to two histidine and an aspartate residues. Preliminary X-ray crystallographic data confirmed that GLX 2-5 is a FeZn protein and contains five histidines, two aspartate, and a bridging water as ligands in the metal binding site. A model for the active site of A. thaliana GLX 2-5 is proposed based on this study.