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Updated: Jul 14, 2026

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
Hemin-mediated para-hydroxylation of aniline: a potential model for oxygen activation and insertion reactions of
Abstract:
The activation of molecular oxygen by alkaline hemin (ferriprotoporphyrin IX) has been studied. In the presence of reductant nicotineamide adenine dinucleotide (NADH) or nicotineamide adenine dinucleotide phosphate (NADPH) and organic substrate, aniline, hemin activates oxygen to the hydroperoxide anion (HO2-) and subsequently mediates insertion of active oxygen into the benzene ring of the substrate to form p-aminophenol, with a high degree of regiospecificity. Oxygen activation does not occur in the absence of aniline. Stoichiometry of the reaction indicates that two electrons are required per molecule of oxygen activated or atom of oxygen inserted into the substrate aromatic ring system. Direct measurement of H2O2 and of the pKa for maximum rate of p-aminophenol formation (11.7 +/- 0.1) indicate participation of the hydroperoxide anion as the active oxygen species in the rate-determining step of the insertion reaction. Powerful scavengers of the hydroxyl radical (OH) have little effect on the formation of H2O2 or p-aminophenol by the system. Superoxide dismutase (10(-7) mol dm-3) inhibited both p-aminophenol and H2O2 formation, when added to the system immediately prior to initiation of the reaction. Studies involving N-phenylhydroxylamine indicate that aromatic ring hydroxylation is occurring directly and not by rearrangement of an N-hydroxylated intermediate. Implications of hemin-mediated hydroxylation reactions for those of enzymatic mixed function oxidase activity are discussed.
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