Biophysical investigation of metal-substituted D-2-hydroxyglutarate dehydrogenase

D-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (EC: 1.1.99.39; Uniprot ID: Q9I6H4) is a metallo-flavoenzyme that utilizes Zn²⁺ and FAD to catalyze the conversion of D-2-hydroxyglutarate to 2-ketoglutarate. The enzyme utilizes Co²⁺, Ni²⁺, Mn²⁺, and Cd²⁺ as alternative metal cofactors. To study how metal substitution impacts flavin properties, the enzyme was purified with different metal ions or treated with EDTA to generate the metallo-apoenzyme (E_FAD-apo). Fluorescence assays revealed distinct metal ion binding sites in the enzyme: concentrations of metal ions up to ∼0.40 mM increased flavin fluorescence at 531 nm, whereas concentrations above ∼0.40 mM quenched flavin fluorescence with a 2-6 nm bathochromic shift. Concomitantly, enzyme-specific activity exhibited a sigmoidal increase, indicating a metal-induced conformational change. CD spectra showed no significant shifts at ∼209 and ∼220 nm but a ≤ 2-fold increase in mean residue ellipticity compared to E_FAD-apo. Metal binding also caused a 2-9 nm bathochromic shift in flavin absorption and emission maxima, indicating stabilization of the excited-state flavin π-electron system. The binding of Zn²⁺, Co²⁺, Mn²⁺, or Cd²⁺ to the enzyme increased by ∼1 unit of the pK_a value of the flavin N₃ atom compared to the E_FAD-apo, consistent with metal-hydrate perturbing flavin electronic properties. In contrast, Ni²⁺ binding decreased the pK_a value, consistent with flavin N₃ atom deprotonating before the Ni²⁺-hydrate in the enzyme active site. These findings reveal that metal ion substitution has minimal impact on the electronic properties of the flavin and the overall structural integrity of the enzyme, highlighting the potential use of metal-substituted variants of the enzyme as biomimetic catalysts.