Glyoxalase 2 is a ␤-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1 H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ؎ 0.15 eq of iron and 1.31 ؎ 0.05 eq of Zn(II) and to exhibit k cat and K m values of 129 ؎ 10 s ؊1and 391 ؎ 48 M, respectively, when using S-D-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Zn(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese.1 H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).The glyoxalase system consists of two enzymes, lactoylglutathione lyase (glyoxalase I, GLX1) 2 and hydroxyacylglutathione hydrolase (glyoxalase II, GLX2), that act coordinately to convert a variety of ␣-ketoaldehydes into hydroxy acids in the presence of glutathione (1). Aromatic and aliphatic ␣-ketoaldehydes react spontaneously with glutathione to form thiohemiacetals, which are converted to S-(2-hydroxyacyl)glutathione derivatives by GLX1. GLX2 hydrolyzes S-(2-hydroxyacyl)glutathione derivatives to regenerate glutathione and produce hydroxy acids. Glyoxalase I, a Zn(II) or Ni(II) metalloprotein, can utilize a number of ␣-ketoaldehydes (2). However, the primary physiological substrate of the enzyme is thought to be methylglyoxal (MG), a cytotoxic and mutagenic compound that is formed primarily as a by-product of carbohydrate and lipid metabolism (3, 4). MG can react with DNA to form modified guanylate residues (5) and interstrand cross-links (6). It also reacts with proteins to form glycosylamine derivatives of arginine and lysine and hemithioacetals with cysteines (7).Cells with high glycolytic rates exhibit high rates of methylglyoxal formation and increased levels of GLX1 activity. For instance, increased levels of GLX1 and GLX2 RNA and protein have been detected in tumor cells, including breast carcinoma cells (8). Because selec...