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

TL;DR

A mutation in an enzyme's active site causes it to react with oxygen, producing superoxide and changing its catalytic behavior.

Contribution

The study shows how a single mutation can induce oxygen reactivity and alter pH profile slopes in flavoproteins.

Findings

The E246L mutation in PaDADH induces oxygen reactivity, producing superoxide during substrate oxidation.

Superoxide reacts with protons in the active site, causing a nonstoichiometric pH profile slope of 1.5.

Adding superoxide dismutase corrects the pH profile slope to 1.0, confirming superoxide's role in the reaction.

Abstract

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•ˉ) during substrate oxidation. The O2•ˉ reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme’s log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•ˉ 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.

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