Biochemical characterization and mutational analysis of the mononuclear non-haem Fe²⁺ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii Available to Purchase

β-Diketone-cleaving enzyme Dke1 is a homotetrameric Fe²⁺-dependent dioxygenase from Acinetobacter johnsonii. The Dke1 protomer adopts a single-domain β-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe²⁺ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure–function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp-binding site for non-haem Fe²⁺, we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe²⁺ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe²⁺) and fluorescence titration (Fe²⁺, Cu²⁺, Ni²⁺, Mn²⁺ and Zn²⁺) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe²⁺ with a dissociation constant (K_d) of 5 μM (site I) and ∼0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe²⁺, but not that of the other bivalent metal ions, at site I, while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe²⁺. The Fe²⁺ catalytic centre activity (k_cat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the k_cat value of 8.5 s⁻¹ for the wild-type enzyme in the reaction with pentane-2,4-dione. The H64N mutant was not catalytically competent, except in the presence of external Fe²⁺ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe²⁺ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe²⁺ into Fe³⁺, which is then released from the metal centre.