Redox Thermodynamics of Dehaloperoxidase-Hemoglobin
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Date
2010-04-09
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Abstract
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 of the free energy
contributions that give rise to this high reduction potential is due to the redox-coupled
conformational change that happens with the distal histidine (H55) between 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. Both binding modes cause the shift in potential to be
negative.
Proximal region mutations were also explored for the purpose of installing in the socalled
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 there being 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 made successful. 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, Xray
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. By establishing the optimum conditions in obtaining
reversible cyclic voltammetry while maintaining surface stability of DHP, this groundwork
will be useful for future studies directed at immobilized DHP electrocatalysis.
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Keywords
Protein Electrochemistry, Dehaloperoxidase, Hemoglobin, Peroxidase, DHP
Citation
Degree
PhD
Discipline
Chemistry
