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