Show simple item record

dc.contributor.authorChand, Subhashen_US
dc.date.accessioned2013-07-22T20:15:13Z
dc.date.available2013-07-22T20:15:13Z
dc.date.issued2013-07-22
dc.date.submittedJanuary 2013en_US
dc.identifier.otherDISS-12120en_US
dc.identifier.urihttp://hdl.handle.net/10106/11879
dc.description.abstractThis study addresses the mechanistic relationships between formation of reactive oxygen species (ROS) and their catalytic oxidation functions in oxygenation and peroxygenation reactions in heme enzymes. Even though both cytochrome P450s (CYP P450s) and peroxidases have different catalytic activities, the involvement of common ROS (Compound 0 and Compound I) have been proposed. Therefore, to understand the generation and activation of peroxide to form ROS, genetically-engineered myoglobin (Mb) mutants were created by incorporating redox-sensitive 3-amino-L-tyrosine (NH2Tyr) or L-3, 4-dihydroxyphenylalanine (DOPA) into its active site. Distal His 64 replaced with redox amino acids mutant Mb showed excellent turnover rates for thioanisole and benzaldehyde oxidation, compared to the wild-type protein. A 9-fold and 81-fold increase in activity, respectively, was observed in the presence of hydrogen peroxide (H2O2). The presence of a redox unnatural amino acid in the active site enhances the rate of compound I (Cpd I) formation and stabilizes it to form one extra H-bond as compared to the wild type (WT) Mb. This increased oxidation activity in mutants offer insights into the role of the distal active site residues which are involved in acid-base catalysis and distal charge relay "pull" effect in peroxide activation and formation of ROS in heme proteins.Furthermore, cyclic voltammetry (CV) and atomic force microscopy (AFM) were used to investigate the importance of active site orientation of an immobilized protein for direct electron transfer (DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on the modified gold electrode surface in a random multilayered fashion, the Ser 3 replaced with NH2Tyr in Mb mutant, was immobilized via a Diels-Alder reaction specific to the NH2Tyr residue to form a homogeneous monolayer. Electrochemical calculations for the number of surface exposed redox-sensitive molecules on the electrode surface (Γ) and heterogeneous rate constant for DET were 1.29 × 10-10 mol cm-2; 2.3 sec-1 for the WT Mb and 1.54 × 10-10 mol cm-2; 1.3 sec-1 for the S3NH2Tyr Mb mutant, respectively. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover frequencies (TOF) around 1.9 × 103 sec-1 (with 87% conversion) for the WT Mb, and 1.5 × 103 sec-1 for the mutant Ser 3-amino-L-tyrosine (S3NH2Tyr) Mb (with 81% conversion). These results indicate that site-directed single monolayer immobilization affords almost the same number of surface exposed Mb active sites as the random multilayer immobilization strategy, though the latter contains a greater number of protein molecules on the electrode surface. The microarray concept development provides novelty to study protein-protein interactions, drug discovery, and biomedical and proteomic research.en_US
dc.description.sponsorshipPerera, Roshanen_US
dc.language.isoenen_US
dc.publisherChemistry & Biochemistryen_US
dc.titleGenetically Modified Myoglobin As A Mimic For Heme Enzymesen_US
dc.typePh.D.en_US
dc.contributor.committeeChairPerera, Roshanen_US
dc.degree.departmentChemistry & Biochemistryen_US
dc.degree.disciplineChemistry & Biochemistryen_US
dc.degree.grantorUniversity of Texas at Arlingtonen_US
dc.degree.leveldoctoralen_US
dc.degree.namePh.D.en_US


Files in this item

Thumbnail
Thumbnail


This item appears in the following Collection(s)

Show simple item record