Cd2+ Substitution Modulates the Rate of Flavin Reduction in D-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1

Abstract Pseudomonas aeruginosa D-2-hydroxyglutarate dehydrogenase (PaD2HGDH; EC 1.1.99.39) is a Zn2+- and FAD-dependent metallo-flavoenzyme that oxidizes α-hydroxy acid substrates through a metal-triggered flavin reduction mechanism. In this work, Zn2+ was replaced by Cd2+ to define how metal identity governs substrate capture, flavin reduction, and conformational gating during catalysis of PaD2HGDH. Relative to the native Zn2+ enzyme, Cd2+ lowered catalytic efficiency for substrate capture (kcat/Km) by ∼10-fold (∼10,000 to 1,000 M–1 s–1) and decreased the rate of flavin-reduction (kred) by ∼5-fold (68 s–1 to 13 s–1), with the Kd value increasing by ∼2-fold. These results are consistent with the larger size of Cd2+, which alters the optimal orientation of the substrate C2 atom relative to the FAD N5 atom. The solvent-viscosity effect indicated that, in the Cd2+ enzyme, kcat, kcat/Km, and kred follow normal hyperbolic viscosity dependences, whereas Kd varies linearly with a slope > 1. This pattern suggests a solvent-sensitive internal isomerization that becomes rate-relevant and conformationally gates both substrate binding and flavin reduction with D-malate as a model substrate. In contrast, the Zn2+ enzyme shows linear viscosity effects consistent with an internal isomerization step that is hidden by slower product release. Together with prior results from Ni2+ substitution, these findings support a unified model in which the divalent metal center not only activates the α-hydroxy acid but also tunes metal–ligand geometry and electrostatics to reshape the ES ⇌ ES* landscape, thereby modulating binding and intramolecular electron transfer to FAD. This mechanistic framework informs metal-dependent flavin reduction and supports the development of metal-chelating inhibitors targeting PaD2HGDH to help address multidrug resistance in P. aeruginosa.