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 (O) 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 (E) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E residue with leucine affects PaDADH catalysis and its ability to react with O using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O reactivity in the EL variant, resulting in a reduced flavin semiquinone species and superoxide (O-) during substrate oxidation. The O- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (k/K) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O- 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.