Bridging the functional gap between reactivity and inhibition in dehaloperoxidase B from Amphitrite ornata: Mechanistic and structural studies with 2,4- and 2,6-dihalophenols.

The multifunctional catalytic globin dehaloperoxidase (DHP) from the marine worm Amphitrite ornata was shown to catalyze the HO-dependent oxidation of 2,4- and 2,6-dihalophenols (DXP; X = F, Cl, Br). Product identification by LC-MS revealed multiple monomeric products with varying degrees of oxidation and/or dehalogenation, as well as oligomers with n up to 6. Mechanistic and O-labeling studies demonstrated sequential dihalophenol oxidation via peroxidase and peroxygenase activities. Binding studies established that 2,4-DXP (X = Cl, Br) have the highest affinities of any known DHP substrate. X-ray crystallography identified different binding positions for 2,4- and 2,6-DXP substrates in the hydrophobic distal pocket of DHP. Correlation between the number of halogens and the substrate binding orientation revealed a halogen-dependent binding motif for mono- (4-halophenol), di- (2,4- and 2,6-dihalophenol) and trihalophenols (2,4,6-trihalopenol). Taken together, the findings here on dihalophenol reactivity with DHP advance our understanding of how these compounds bridge the inhibitory and oxidative functions of their mono- and trihalophenol counterparts, respectively, and provide further insight into the protein structure-function paradigm relevant to multifunctional catalytic globins in comparison to their monofunctional analogs.