The proposal extends previous studies on utilizing the oxyferryl heme center of cytochrome c peroxidase (CCP) for the oxidation of novel small molecule substrates. Previous work focused on how the protein environment defines the physical, spectroscopic and functional properties of heme peroxidases. These suggested that oxidative reactions catalyzed by some heme enzymes might be recruited into the scaffold of others. Significant progress has been made in the last grant period on efforts to introduce small molecule binding sites into the enzyme by cavity complementation. These results illustrate the feasibility of this approach for placing potential substrates near the heme active site and in addition, they have provided a framework for answering some more general questions about weak interactions in ligand-protein complexes and surface loop movements that were not originally anticipated. In the next funding cycle the Principal Investigator seeks to refine and extend these concepts to focus to an increasing degree on using the information obtained earlier about the factors of the protein environment that control the energetics, reactivity, and specificity of the active site, in order to introduce novel substrate binding into the enzyme and to characterize these interactions and their potential for being oxidized by the heme. Two general lines of inquiry will be proposed for the next period- 1) what can these artificial binding sites tell us about heme enzyme function and protein-ligand interactions, and 2) can these sites be used to engineer enzymes with novel function? More specifically, in the first area, the Principal Investigator will explore the more general aspects of creating artificial cavities in CCP and characterizing the specificity, kinetics and energetics associated with small molecule binding to these sites. The second emphasis will draw upon this work to examine the oxidative chemistry that may occur at sites that are placed at different positions with respect to the heme. These studies may help provide an increased understanding of what factors control the very different activities exhibited by peroxidases on one hand, which oxidize substrates by electron or hydrogen atom transfer, and monooxygenases on the other, which operate by ferryl oxo-transfer chemistry. Thus, these artificial enzymes may help provide a better understanding of the function of P450, nitric oxide synthase, indoleamine 2,3-dioxygenase, and prostaglandin synthase, each of which have significant medical importance.
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