The binding of iron and zinc to glyoxalase II occurs exclusively as di-metal centers and is unique within the metallo-β-lactamase family

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Abstract
Cytosolic glyoxalase 2 (GLX2-2) from Arabidopsis thaliana is a metalloenzyme that has been shown to bind a mixture of Zn, Fe, or Mn when produced in cells grown in rich media. In an effort to prepare metal-enriched samples, GLX2-2 was over-expressed in minimal media containing either Zn, Fe, or Mn. The resulting enzymes bound an average of 1 equivalent of metal ion and were partially enriched with a specific metal ion. The enzymes produced in minimal media were active towards the substrate S-d-lactoylglutathione, yielding k cat/K m values similar to those of rich media GLX2-2. EPR studies on minimal media GLX2-2 samples revealed spectra which were identical to those over-expressed in rich media that contained nearly 2 equivalents of metal. The EPR spectra showed the presence of antiferromagnetically and ferromagnetically coupled, dinuclear metal centers. EXAFS spectra on the minimal media GLX2-2 samples over-expressed in the presence of Fe or Zn were also very similar to those of the rich media GLX2-2 samples, indicating the presence of dinuclear metal centers. The EXAFS studies also demonstrate that Zn(II) and Fe (in the Fe-enriched sample) are distributed in the dinuclear site. These data indicate that the minimal media GLX2-2 samples are a mixture of fully loaded, dinuclear metal-containing enzyme and metal-free enzyme. This characteristic of A. thaliana GLX2-2 makes it unique among the other members of the metallo-β-lactamase family in that it does not ever appear to exist as a mononuclear metal ion containing enzyme and that it exhibits positive cooperativity in metal binding.