NOVEL SUBSTRATE OXIDATION BY ENZYME ENGINEERING

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The proposed studies will explore the potential of utilizing the oxyferryl heme center of cytochrome c peroxidase (CCP) for the oxidation of novel small molecule substrates. Two approaches will be taken to develop this concept: The construction a number of artificial variants and hybrid enzymes that may confer oxidizing activity toward a variety of small molecules, and a detailed investigation of the structural and biophysical effects that these and other alterations have upon the heme active site. Such artificial enzymes will have relevance to such biologically important catalysts as P450, nitric oxide synthase, indoleamine 2,3-dioxygenase, horseradish peroxidase, and manganese dependent ligninase. Throughout these studies, a central and unresolved question will be addressed: How does the protein environment direct the chemistry of the oxidized heme toward such diverse reactions as alkene epoxidation and hydroxylation, intramolecular electron transfer and radical chemistry, and intermediary metal ion oxidation? The following specific aims will be addressed: 1) The role that the protein environment plays in controlling the redox properties of CCP will be examined. 2) The effects on the heme electronic structure resulting from modulating the metal-ligand interaction and coordination state will be studied. 3) the interaction of the Trp-191 free radical and alternative radical centers to the heme will be explored. 4) Artificial substrate sites will be introduced into the proximal and distal heme environment, and these proteins will be examined for novel substrate oxidation. 5) Portions of other peroxidases will be grafted into the sequence of CCP to introduce a broad range of substrate oxidizing capabilities into this structure. 5) Each of these enzymes will be structurally characterized to guide further design efforts. The variations in reactivity, metal- ligand binding, electron transfer efficiency, and redox potential of heme enzymes are due in part to the sensitivity of the metal electronic valence states to subtle effects of covalency and chemical environment. However, even if a well defined structural change has been introduced, it is very difficult as yet to predict the consequences that such a change in structure will have on the metal electronic energy levels and thus on the function of the protein. For these reasons it is important to study not just the structural alteration of these enzymes and their functional consequences, but to measure and relate directly the ensuing changes in the properties of the metal electronic structure.