Description: Background: Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, alpha and beta, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact alpha-site and conserved beta-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative beta-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion. Results: Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the beta-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 A structure of the double mutant confirmed that the beta-site was eliminated but that the active site remained otherwise intact. Conclusions: Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.