Spectroscopic Studies of Activation of Dehaloperoxidase by Hydrogen Peroxide

Abstract

ABSTRACT

ZAO ,WANG. Spectroscopic Studies of Activation of Dehaloperoxidase by Hydrogen Peroxide. ( Under the direction of Tatyana I. Smirnova and Stefan Franzen.)

The focus of this research is to use stopped-flow UV-visible and rapid freeze-quench electron paramagnetic resonance (EPR) spectroscopic methods to study the peroxidase activity of the enzyme dehaloperoxidase (DHP) from Amphitrite ornata. DHP is the first characterized hemoglobin which has a naturally occurring peroxidase function. It has been discovered that ferric DHP can catalyze the dehalogenation of halophenol to quinones in the presence of H2O2, which is a peroxidase function. However, ferrous DHP is also capable of binding oxygen reversibly, which is a hemoglobin function. It is still not understood how this bi-functional protein can act as both a hemoglobin and a peroxidase, because hemoglobin function require ferrous form as starting oxidation state but peroxidase require ferric form as starting oxidation state. This work has addressed three separate questions. First, I studied the autoreduction reaction of DHP under CO atmosphere. The study demonstrates that the Cys 73 residue is not required for autoreduction of DHP under CO atmosphere. Mutation of Cys 73 to serine also does not affect the mechanism of the DHP reaction with hydrogen peroxide. Second, the study demonstrates that in the absence of a substrate, DHP reacts with hydrogen peroxide forming Compound II, that, under conditions used, decays after 2 seconds to form Compound RH. In the presence of the substrate, TCP (2,4,6-trichloropenol) Compound II reacts with TCP forming Ferric DHP and DCQ (2,4-dichloroquinone).