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

• DOI: 10.1111/febs.16591
• Publication Date: 2022-08-09T07:14:38Z
• Authors: Panu Pimviriyakul; Pimchai Chaiyen
• Keywords: Hydroxylation

HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation denitration respectively. C4a-hydroperoxy-FAD is a key reactive intermediate wherein its formation, protonation stabilization reflect enzyme efficiency. Herein, transient kinetics, site-directed mutagenesis pH-dependent behaviours reaction were employed to identify features stabilizing C4a-adducts in HadA. The formation pH independent, whereas decay distal oxygen are associated with pKa values 8.5 8.4 These correlated product within range 7.6-9.1, indicating importance adduct enzymatic We identified Arg101 as residue for reduced FAD (FADH- ) binding due loss these abilities well activity HadAR101A HadAR101Q . Mutations neighbouring Asn447 do not affect rate formation; however, they impair FADH- binding. disruption Arg101/Asn447 hydrogen bond networking HadAN447A increases value 9.5; this was altered HadAN447D (pKa 8.5). Thus, pair should provide important interactions maintain H2 O2 elimination from In presence substrate, C4a-hydroxy-FAD at hydroxylation step insensitive, it dehydrates form oxidized 7.9. This structural feature might help elucidate how stabilized other flavin-dependent monooxygenases.