Through the research program defined in this application, we seek to understand the molecular mechanisms of metalloenzyme function and the relationship of metal coordination to the specific activities of various metalloproteins. Our first efforts will be focused on the oxygen binding and metabollic proteins hemoglobin, myoglobin, peroxidase, and cytochrome P-450. Protein systems with metal cofactors are ubiquitous in the essential biotransformations of most living tissues and are associated with many well defined disease states. In many cases, the metal center offers a unique catalytic activity which is dictated by the metal coordination sphere. We propose to investigate the precise role of the metal ligands in various catalytic process through the use of site specific mutagenesis and other modern recombinant DNA techniques to specifically alter this ligation sphere. We will then determine the effects of such modifications on the structure and activity of the metalloprotein. The overall goal is to separate those features and reactivities which are due solely to the coordinating ligands and those which are dictated by the protein structure and environment. Emerging from these investigations will be the first clear documentation of the role of metal coordination in the biological activity of an important group of metalloprotein systems. Our first specific aim will focus on the cytochrome P-450 monoxygenase systems found ubiquitously in nature and playing key roles in detoxification, xenobiotic metabolism, and steroid biosynthesis. The cytochromes P-450 possess the unique chemical reactivities associated with unactivated alkane hydroxylation, and also contain a likewise uncommon thiolate axial ligand to the heme center. We wish to determine the function of this cysteine ligand in dictating the relevant chemical reactivities and biophysical properties of the P-450 oxygenases. Recombinant DNA technology will be used to change the heme axial ligand in cytochrome P-450 from cysteine to the histidine side chain more commonly found in myoglobin, hemoglobin, and peroxidase. The converse experiment will replace the histidine residue in peroxidase and hemoglobin/myoglobin with a thiolate. Through application of EPR, ENDOR, Mossbauer, Raman, MCD and X-ray structure determination, along with rapid reaction and precise chemical reactivity documentations, it will be possible to clearly separate the contributions of axial ligand, specific active site groups, and protein environment to the unique electron transfer and oxygen chemistries associated with the P-450 heme center.
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