Spectroscopic Characterization of H93G Myoglobin Cavity Mutant As A Versatile Protein Scaffold For Modeling Native Heme Iron Coordination Structures and Investigation of the Structure and Mechanism of the Dual-Function Enzyme, Amphitrite Ornata Dehaloperoxidase
Date of Award
Campus Access Dissertation
Chemistry and Biochemistry
John H. Dawson
His93Gly sperm whale myoglobin (H93G Mb) 'cavity' mutant, having the proximal histidine ligand removed, is a versatile template for establishing the structure of the heme iron coordination unit in proteins. H93G Mb complexes with different exogenous ligands have been successfully prepared as models for native heme iron active sites ligated by proximal His (imidazole), Lys (amines), seleno-Cys (selenols) and Met (thioethers). Particularly, a variety of accurate ambient-temperature mixed-ligand model complexes, hard to achieve with synthetic models in organic solvents, have been successfully prepared by utilizing H93G Mb. All of the newly prepared H93G Mb adducts have been characterized with magnetic circular dichroism and UV-visible absorption spectroscopy. The resulting data have substantially expanded the spectral database which is utilized to establish the coordination structure of heme centers in newly discovered heme proteins of unknown structure at the metal unit.
Amphitrite ornata dehaloperoxidase (DHP) is the first heme-containing globin possessing a biologically relevant peroxidase enzymatic activity. DHP catalyzes the H2O2-dependent detoxifying dehalogenation of halophenols in their living environment. Although DHP is isolated in the catalytically inactive oxyferrous state, we find that, in the presence of 2,4,6-trichlorophenol substrate, a stoichiometric amount of H2O2 converts oxyferrous DHP to the enzymatically active ferric state. In addition, we have successfully generated and characterized several active site mutants of DHP and Mb. The "peroxidase-like" Mbs exhibit increased dehaloperoxidase activity while the "Mb-like" DHP mutant has attenuated DHP activity. The results contribute evidence supporting the proposal that DHP evolved from a dioxygen carrier globin protein and therefore possesses dual physiological roles of O2 carrier and dehaloperoxidase. The high peroxidase activity in distal-site blocker L100F DHP mutant demonstrates that substrate binding in the distal cavity is not a prerequisite for DHP function. These current studies provide the scientific foundation for a new structure-function paradigm for peroxidases and for the genetic evolution of the dual-function enzyme DHP.
Du, J.(2010). Spectroscopic Characterization of H93G Myoglobin Cavity Mutant As A Versatile Protein Scaffold For Modeling Native Heme Iron Coordination Structures and Investigation of the Structure and Mechanism of the Dual-Function Enzyme, Amphitrite Ornata Dehaloperoxidase. (Doctoral dissertation). Retrieved from http://scholarcommons.sc.edu/etd/677