Redox Thermodynamics of Dehaloperoxidase-Hemoglobin

Protein Electrochemistry;Dehaloperoxidase;Hemoglobin;Peroxidase;DHP

D'Antonio, Edward Lawrence ; Dr. Robert B. Rose, Committee Member,Dr. Edmond F. Bowden, Committee Chair,Dr. Stefan Franzen, Committee Member,Dr. David C. Muddiman, Committee Member,D'Antonio, Edward Lawrence ; Dr. Robert B. Rose ; Committee Member ; Dr. Edmond F. Bowden ; Committee Chair ; Dr. Stefan Franzen ; Committee Member ; Dr. David C. Muddiman ; Committee Member

University:North Carolina State University

关键词: Protein Electrochemistry;    Dehaloperoxidase;    Hemoglobin;    Peroxidase;    DHP;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/6189/etd.pdf?sequence=1&isAllowed=y

美国|英语

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【 摘 要 】

Dehaloperoxidase-hemoglobin (DHP) is a small intracellular hemoglobin found in theterebellid 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 betweenthe globin and peroxidase classes of heme proteins, various properties of DHP have beenfound to be unique. Among one of these properties is the Fe(III)/Fe(II) formal reductionpotential, which has been determined herein, in solution and surface-bound. Furthermore,electrochemical investigations of DHP have not been explored to any significant extent. Theformal reduction potential of DHP is much more positive than any known peroxidase andmore positive than any intracellular globin. A thermodynamic analysis of the free energycontributions that give rise to this high reduction potential is due to the redox-coupledconformational 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 distalbinding pocket and it can bind substrates, such as 2,4,6-trihalophenols on the external side ofthe protein. When DHP is exposed to these halophenols, it was determined that a modulationin the Fe(III)/Fe(II) oxidation/reduction potential occurs and the result is more substantial forinternal binding than external. Both binding modes cause the shift in potential to benegative.Proximal region mutations were also explored for the purpose of installing in the socalledAsp-His-Fe triad into DHP, which is generally found in peroxidases but not globins, sothat the “pushâ€effect could be studied. The “pushâ€effect refers to there being anioniccharacter on the proximal ligand of a heme peroxidase, which has a role in “pushingâ€apartthe O-O bond of hydrogen peroxide in the peroxidase reaction. So far a globin model systemhas not been made successful. These results show that by making this type of mutation intoDHP (i.e. the M86D mutation), the mutant causes H55 to coordinate as the sixth ligand to theiron atom and inhibits all peroxidase activity, under physiological conditions. The studyclarifies that globins simply do not have this structural feature because they are not designedto carry out peroxidase chemistry. Electrochemical results aided in characterizing if thesemutants had established the triad. Other structural techniques employed were 13C-NMR, Xraycrystallography, and resonance Raman spectroscopy.Finally, an electrochemical study of the Fe(III)/Fe(II) redox couple of DHP adsorbedto a self-assembled monolayer surface on a gold working electrode was carried out formethod development purposes. By establishing the optimum conditions in obtainingreversible cyclic voltammetry while maintaining surface stability of DHP, this groundworkwill be useful for future studies directed at immobilized DHP electrocatalysis.