| Dehaloperoxidase (DHP), a hemoglobin from the marine worm Amphitrite ornate, not only binds and transports dioxygen reversibly, but also has evolved a peroxidase-like enzymatic function for detoxifying halogenated organic compounds released by co-habitant annelids. DHP catalyzes the oxidative dehalogenation of 2,4,6-trihalophenols to 2,6- dihaloquinones in the presence of H2O2. Underlying this bifunctionality is an unusual distal histidine (H55) that is displaced ∼ 1 A farther from the heme iron than distal histidines in other globins, leading to a decreased interaction with distal ligands (O2, H2O) through hydrogen bonding. As a result, DHP's globin function (dioxygen binding) and enzyme function (dehalogenation), wherein H55 serves as an acid-base catalyst, are both impacted. Additionally, H55 shows an unusual flexibility leading to the conventional 6-coordinate "closed" conformation common to other globins and a unique 5-coordinate "open" conformation. Although increasingly extensive spectroscopic and crystallographic characterization of DHP have become available in recent years, electrochemical measurements remain limited, for the most part, to reports of ferric/ferrous reduction potentials of DHP and its mutants. The broad aims of the present work are to develop the requisite voltammetric methodology to enable electrochemical characterization of DHP in diffusional and diffusionless formats and to then utilize this methodology in the determination of kinetic, thermodynamic, and mechanistic properties for its ferric/ferrous reaction under both anaerobic and aerobic conditions.;Diffusional cyclic voltammetry (CV) of DHP was achieved on mixed-self-assembled monolayer (SAM) composed of hydroxyl- and carboxyl-terminated alkanethiols on gold along with stringent protein purification procedures. Anaerobic conditions gave rise to stable and reproducible voltammetry featuring quasi-reversible electron transfer (ET) kinetic behavior. ET to ferric DHP is postulated to proceed through the "open" conformational state, which exists in equilibrium with the "closed" state at ambient temperature. Under aerobic conditions, chemically reversible CV was achieved for DHP, an observation unprecedented in the globin electrochemical literature. The presence of ambient dioxygen shifted the ferric/ferrous reduction potential positive by ∼0.1 V. An electrochemical-chemical (EC) reaction mechanism was proposed, and with the aid of computer simulation, thermodynamic and kinetic parameters for dioxygen binding to deoxyferrous DHP (II) were obtained. Compared to sperm whale myoglobin, oxygen binding in DHP was found to be weaker (Kb = 0.38 vs 1.2 muM-1) and more labile (k off = 1.14 x 104 vs 12 s-1). The enhanced chemical reversibility of DHP voltammetry under aerobic conditions appears to be largely due to the larger oxygen dissociation rate constant (koff). It is believed that DHP's unique distal histidine is largely responsible for these unique kinetic and thermodynamic properties.;Diffusionless CV was characterized for adsorbed DHP using the same type of mixed- SAM electrode for a variety of solution conditions (pH, ionic strength, electrolyte composition). The adsorption of DHP was demonstrated to be chemically reversible and dominated by electrostatic forces. Cyclic voltammetric measurement of electroactive surface concentration (Gammasurf) revealed an inverse scan rate dependence wherein higher values of Gammasurf resulted from lower scan rates. This observation was explained by invoking a dynamic docking model previously developed for ET between myoglobin and cytochrome b5..;Two preliminary studies were conducted intending to improve the extent and stability of immobilized DHP. Covalent immobilization of DHP on the mixed-SAM electrode was demonstrated. Improved stability was observed, however, the system suffered from low Gammasurf values. Five surface mutants designed to enhance positive charge in the vicinity of the hemeedge were prepared and evaluated by CV. Improvement in stability and extend of Gammasurf and ET kinetics were observed and discussed with respect to mutation locations and the overall surface charge. Based on these initial results, potential future mutations were suggested. These insights may contribute toward the development of DHP-based biosensors. |
