An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O₂) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E²⁴⁶) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E²⁴⁶ likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E²⁴⁶ residue with leucine affects PaDADH catalysis and its ability to react with O₂ using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O₂ reactivity in the E²⁴⁶L variant, resulting in a reduced flavin semiquinone species and superoxide (O₂^•-) during substrate oxidation. The O₂^•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (k_cat/K_m) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O₂^•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.