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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 2 ) 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 246 ) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E 246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E 246 residue with leucine affects PaDADH catalysis and its ability to react with O 2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O 2 reactivity in the E 246 L variant, resulting in a reduced flavin semiquinone species and superoxide (O 2 • -) during substrate oxidation. The O 2 • - 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 O 2 • - 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., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)