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

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 pK_a 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 HadA_R101A and HadA_R101Q . 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 HadA_N447A increases the pK_a value of C4a-hydroperoxy-FAD decay to 9.5; however, this pK_a was not altered in HadA_N447D (pK_a of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH^- binding and maintain the pK_a associated with H₂ O₂ 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 pK_a of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin-dependent monooxygenases.