The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni2+ ion, whereas the other contains two inactivating Zn2+ ions. Enzymological experiments prompted by the binuclear Zn2+ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn2+/Co2+ -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.