Xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) is a non-heme mononuclear Fe(II) enzyme that decarboxylates alphaKG to succinate and CO(2) while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17% of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K(d) of alphaKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of alphaKG. The N358A variant exhibited a 23-fold decrease in k(cat)/K(m) compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in K(i)(app) of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity.