DETERMINANTS OF FUNCTIONAL DIVERSITY IN HEME ENZYMES

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There is a significant interest in many areas of biological science in developing our abilities to alter or "engineer" proteins aNd enzymes by artificial means. It is often found, however, that we are attempting to modify structures that we do not fully understand, even for cases in which the structure of the protein is precisely known. Before protein manipulation can be reasonably referred to as protein engineering, it is necessary to achieve an understanding, within a group of highly related proteins, of how and to what degree the amino acids surrounding a catalytic site contribute to the properties that distinguish one activity from another. The long term goal of these studies is to be able to convert the properties of one of the heme peroxidases to those of another by alteration of substrate specificities, or to those of another class of heme protein such as myoglobin by alteration of the protein environment of the heme active site. However, many of the structural factors that influence heme enzyme function are still controversial and require closer examination. Therefore, the specific aims of this proposal are to gain a better understanding of the protein interactions that determine functional diversity in these proteins at the atomic level. Functional and spectroscopic properties of variant forms of cytochrome c peroxidase and horseradish peroxidase produced by site-directed mutagenesis will be examined in order to address the following issues. 1. Mutations of cytochrome c peroxidase in the active site channel that have markedly different function will be examined by low temperature electron paramagnetic resonance. This will provide the energy splittings of the low-lying excited states of the Fe+3, and should be a very sensitive method to explore the environmental and conformational effects of mutations on the Fe+3 center. 2. Specific residues positioned between the cytochrome c peroxidase-cytochrome c complex will be altered. Electron transfer measurements will be examined to determine criteria for efficient electron transfer. 3. Studies of the reduction potential of mutationally altered cytochrome c peroxidases may determine whether the very negative potentials observed for peroxidases are due to a single factor, or to a combination of protein environment effects. 4. Studies of cytochrome c peroxidase containing specific isotopic substitutions will broaden a search for the location of the stable free radical compound I intermediate. In addition, the role that amino acid residues near the active site play a controlling the interaction between the heme iron and the free radical site will be investigated. 5. The gene for horseradish peroxidase C will be synthesized and expressed in E. coli to allow comparative mutagenesis studies now in progress with cytochrome c peroxidase.