Structural Studies of Inhibitor and Substrate Binding in the Hemoglobin Dehaloperoxidase

Abstract

Dehaloperoxidase (DHP) is a dual function heme protein found in the marine polychaete Amphitrite ornata. A. ornata is filter feeding worm that inhabits estuary inlets alongside other annelids such as Notomastus lobatus and Thelepus crispus, which secrete various haloaromatics theoretically as a means of territorial protection. N. lobatus, in particular, expels mono-, di-, and trihalogentated phenols. Even though DHP is one of two hemoglobins found in A. ornata, the protein possesses significant peroxidase activity and is capable of oxidatively dehalogenating certain halophenols found in its environment. The ability of DHP to bind monohalogenated phenols in an internal distal cavity separates the protein from any other known globin. A variety of spectroscopic and enzymatic techniques have been utilized to probe halophenol binding in DHP. In order to perform these techniques, codon optimization of the DHP gene was first performed. 1H NMR experiments on low-spin metcyano DHP revealed separate modes of binding between mono-, di-, and trihalogentated phenols. Specifically it was found that binding of mono- and dihalogenated phenols occurs in the internal binding pocket, while trihalogenated phenols bind at a second, external site. This led to enzymatic studies that revealed inhibition of DHP peroxidase activity upon monohalogenated phenol binding in the internal pocket. NMR experiments on the protein backbone of 13C and 15N labeled DHP show trihalogenated substrates induce chemical shift deviations in the distal histidine H55 NεH and amide protons near tryptophan 120. This indicates binding of trihalophenols may occur on the external side of H55 resulting in allosteric changes at the dimer interface, or that binding may occur directly at W120.