Formation and stabilization of C4a‐hydroperoxy‐FAD by the Arg/Asn pair in HadA monooxygenase

• Published in: The FEBS journal 2023-01, Vol.290 (1), p.176-195
• Main Authors: Pimviriyakul, Panu, Chaiyen, Pimchai
• Format: Article
• Language: English
• Subjects: Adducts; Binding; C4a‐hydroperoxyflavin; Decay; Dehalogenation; Dehydration; Detoxification; Enzymatic activity; Enzyme activity; Enzymes; Flavin; Flavin-adenine dinucleotide; Flavin-Adenine Dinucleotide - metabolism; Flavins - metabolism; flavin‐dependent monooxygenase; halogenated phenol; Hydrogen bonds; Hydrogen peroxide; Hydroxylation; Kinetics; Mixed Function Oxygenases - metabolism; Mutagenesis, Site-Directed; Mutation; nitrophenol; Oxidation-Reduction; pH effects; Phenols; pH‐dependent; Protonation; Site-directed mutagenesis; Stabilization; Substrates
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.16591

HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation and denitration respectively. C4a‐hydroperoxy‐FAD is a key reactive intermediate wherein its formation, protonation and stabilization reflect enzyme efficiency. Herein, transient kinetics, site‐directed mutagenesis and pH‐dependent behaviours of HadA reaction were employed to identify key features stabilizing C4a‐adducts in HadA. The formation of C4a‐hydroperoxy‐FAD is pH independent, whereas its decay and protonation of distal oxygen are associated with pKa values of 8.5 and 8.4 respectively. These values are correlated with product formation within a pH range of 7.6–9.1, indicating the importance of adduct stabilization to enzymatic efficiency. We identified Arg101 as a key residue for reduced FAD (FADH−) binding and C4a‐hydroperoxy‐FAD formation due to the loss of these abilities as well as enzyme activity in HadAR101A and HadAR101Q. Mutations of the neighbouring Asn447 do not affect the rate of C4a‐hydroperoxy‐FAD formation; however, they impair FADH− binding. The disruption of Arg101/Asn447 hydrogen bond networking in HadAN447A increases the pKa value of C4a‐hydroperoxy‐FAD decay to 9.5; however, this pKa was not altered in HadAN447D (pKa of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH− binding and maintain the pKa associated with H2O2 elimination from C4a‐hydroperoxy‐FAD in HadA. In the presence of substrate, the formation of C4a‐hydroxy‐FAD at the hydroxylation step is pH insensitive, and it dehydrates to form the oxidized FAD with pKa of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin‐dependent monooxygenases. C4a‐hydroperoxy‐FAD is a key reactive intermediate in HadA monooxygenase which catalyses the detoxification of halogenated phenols and nitrophenols. In this work, transient kinetic experiments and structure‐based site‐directed mutagenesis were performed. The pKa associated with individual steps in HadA mechanism were elucidated. Thus, the Arg101/Asn447 pair plays an important role in the formation, stabilization and controlling of the protonation status of C4a‐hydroperoxy‐FAD in HadA which reflect enzyme efficiency.