INSIGHTS INTO THE EVOLUTIONAL ADAPTATIONS AND FUNCTIONAL SWITCHING MECHANISM IN DUAL FUNCTION HEMOGLOBIN/DEHALOPEROXIDASE (DHP) AND MODELING OF CYTOCHROME P450 ACTIVE SITE WITH H93G MYOGLOBIN CAVITY MUTANT
Date of Award
Open Access Thesis
Chemistry and Biochemistry
John H. Dawson
The coelomic hemoglobin, dehaloperoxidase (DHP), from the sea worm Amphitrite ornata, is a dual function oxygen-binding heme protein that also possesses a significant peroxidase activity. As evolved from an ancient oxygen carrier globin, several structural adaptations have enabled DHP to evolutionally gain significantly enhanced peroxidase capability for self-protection while only minimally compromising its primary function as an O2 carrier. To elucidate these adaptations, the peroxidase activities and O2 affinities of several DHP and sperm whale myoglobin (Mb) mutants have been prepared. Several heme environmental structural factors that regulate the dual functions of DHP have been found, providing insight into how DHP has gained significantly enhanced enzymatic capability in an evolutional process for protection from its toxic living environment without compromising its primary function as an O2 carrier. As to the question how DHP interconverts the different oxidation states of heme required for its hemoglobin (FeII) and peroxidase (FeIII) functions, a functional switching mechanism of DHP has been proposed and carefully tested. Using stopped-flow methodology, the H2O2-mediated conversion of oxy-Fe(II) DHP to the ferric state triggered by both biologically relevant (TCP etc.) and non-relevant (ferrocyanide, etc.) compounds was examined. It has been found that ferric heme ligands and spin-trapping reagents can completely inhibit the TCP-triggered DHP functional switch, strongly supporting a proposed mechanism involving substrate radicals (TCP⋅).
To thoroughly study the structures and the functions of the diverse heme enzymes, numerous heme iron model systems have been developed. Using the H93G Mb cavity mutant, pioneered by Barrick, our laboratory has successfully prepared several model heme complexes of defined structure. However, previous attempts to generate H93G adducts with spectral properties resembling those of ferrous thiolate-ligated heme proteins such as cytochrome P450 (P450) and C. fumago chloroperoxidase (CPO), were not successful. In the second part of the dissertation, the first H93G Mb spectral models for ferrous P450 and CPO have been generated with phosphine as the axial ligand. The novel heme-phosphine complexes have been comprehensively characterized in their ferric, ferrous and ferryl states with UV-visible absorption and MCD spectroscopy. The data provide important insight into the unique spectral properties of thiolate-ligated heme iron systems and further expand our extensive H93G Mb spectral model library.
Sun, S.(2014). INSIGHTS INTO THE EVOLUTIONAL ADAPTATIONS AND FUNCTIONAL SWITCHING MECHANISM IN DUAL FUNCTION HEMOGLOBIN/DEHALOPEROXIDASE (DHP) AND MODELING OF CYTOCHROME P450 ACTIVE SITE WITH H93G MYOGLOBIN CAVITY MUTANT. (Master's thesis). Retrieved from https://scholarcommons.sc.edu/etd/2906