Redox thermodynamics of dehaloperoxidase-hemoglobin

Dehaloperoxidase-hemoglobin (DHP) is a small intracellular hemoglobin found in the terebellid polychaete Amphitrite ornata. This heme protein can transport oxygen to the cells, but it also has moderate peroxidase activity. As a result of its hybrid functionality between the globin and peroxidase classes of heme proteins, various properties of DHP have been found to be unique. Among one of these properties is the Fe(III)/Fe(II) formal reduction potential, which has been determined herein, in solution and surface-bound. Furthermore, electrochemical investigations of DHP have not been explored to any significant extent. The formal reduction potential of DHP is much more positive than any known peroxidase and more positive than any intracellular globin. A thermodynamic analysis indicates the free energy contributions that give rise to this high reduction potential is due to the redox-coupled conformational change from interactions between the distal histidine (H55) and the Fe(III) and Fe(II) oxidation states.;DHP is also known to bind inhibitors, such as para-halophenols, inside its distal binding pocket and it can bind substrates, such as 2,4,6-trihalophenols, on the external side of the protein. When DHP is exposed to these halophenols, it was determined that a modulation in the Fe(III)/Fe(II) oxidation/reduction potential occurs and the result is more substantial for internal binding than external binding. Both binding modes cause the shift in potential to be negative.;Proximal region mutations were also explored for the purpose of installing the so-called Asp-His-Fe triad into DHP, which is generally found in peroxidases but not globins, so that the "push" effect could be studied. The "push" effect refers to anionic character on the proximal ligand of a heme peroxidase, which has a role in "pushing" apart the O-O bond of hydrogen peroxide in the peroxidase reaction. So far a globin model system has not been successfully converted into a peroxidase through the addition of the proximal aspartate residue. These results show that by making this type of mutation into DHP (i.e. the M86D mutation), the mutant causes H55 to coordinate as the sixth ligand to the iron atom and inhibits all peroxidase activity, under physiological conditions. The study clarifies that globins simply do not have this structural feature because they are not designed to carry out peroxidase chemistry. Electrochemical results aided in characterizing if these mutants had established the triad. Other structural techniques employed were 13C-NMR, X-ray crystallography, and resonance Raman spectroscopy.;Finally, an electrochemical study of the Fe(III)/Fe(II) redox couple of DHP adsorbed to a self-assembled monolayer surface on a gold working electrode was carried out for method development purposes. We established the optimum conditions for obtaining reversible cyclic voltammetry while maintaining surface stability of DHP. This groundwork will be useful for future studies directed at immobilized DHP electrocatalysis.