Making Substrates Out of Inhibitors: Distal Cavity Mutations in Dehaloperoxidase from Amphitrite Ornata

Abstract

Dehaloperoxidase (DHP A), a unique multi-function enzyme, from the marine worm Amphitrite ornate dehalogenates 2,4,6-trihalophenols to form the corresponding 2,6-dihalogenated quinone product. The catalytic cycle of DHP is similar to horseradish peroxidase (HRP), involving a high-valent oxoferryl heme and two one electron transfers from the aromatic substrate to the enzyme. However, unlike HRP, DHP has an internal binding cavity on the distal side of the heme capable of accommodating monohalogenated phenols. Internal binding of monohalogenated phenols in the distal cavity of DHP inhibits peroxidase function. Therefore, even though the peroxidase mechanism of DHP is similar to HRP, DHP is not capable of oxidizing the same range of halogenated substrates. Blocking internal binding in DHP may be the key to effectively enable DHP to function as a peroxidase on the full range of halogenated phenols. The distal cavity of DHP is surrounded by several hydrophobic amino acids that stabilize internal binding of the monohalogenated phenols: several phenylalanine residues (F21, F24, and F35), a valine residue (V59), and a leucine residue near the back edge of the heme (L100). We have recently expressed the L100F, L100Q, L100T, and L100N mutants of DHP in an effort to prevent internal binding and turn the inhibitors into substrates. These mutants are being characterized by UV-vis spectroscopy, resonance Raman spectroscopy, X-ray crystallography, and molecular dynamics simulations. Kinetic assays indicate that the peroxidase activity of the L100 mutants is reduced compared to native DHP, and although we believe it to be possible theoretically, none of the L100 mutations have caused the switch from inhibitor to substrate up to the present.