P450BM-3--MECHANISM OF ELECTRON TRANSFER 02 ACTIVATION

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The long range goal of this research project is to elucidate the mechanism of electron transfer and substrate and oxygen activation by cytochrome P- 450-type enzyme systems. P-450 enzymes systems can be divided into two general classes; 1) those which are have only two protein components in their electron transfer system: an NADPH-P-450 reductase and the P-450; and, 2) those which have three proteins in their electron transfer system: a flavoprotein-iron sulfur protein reductase, an iron sulfur protein ,and the P-450 is relatively specific. In eucaryotes, these enzymes are found in the microsomal and mitochondria fractions respectively. While much is known about protein/protein interaction in the mitochondrial-type system, as a consequence of the vast amount of work which has been done with both P-450cam and P-450scc, there is relatively little precise structural information regarding the very important microsomal drug metabolizing enzymes because of the lack of a comparable soluble enzyme on which to conduct model studies. The recent isolation of a soluble P-450 (P-450BM-3) from Bacillus megaterium containing both the P-450 reductase and the P-450 on a single polypeptide chain and the establishment of the similarities between this enzyme and mammalian microsomal P-450 systems was very exciting. This protein should serve as the model system to clarify structural and mechanistic questions regarding these important drug metabolizing enzymes. With the recent cloning and expression of P-450BM-3, this enzymes is now available in gram quantities in homogeneous form. The Specific Aims of this project are to determine: 1) the kinetics and thermodynamics of the reactions of the individual redox active centers of this multidomain protein; 2) the factors which control protein/protein interaction and domain recognition during electron transfer and oxygen activation; and, 3) the three-dimensional structure of this protein using x-ray crystallography. The first phase will divide (physically and kinetically) the domains of the protein into the reductase and P-450 regions and compare the properties of these domains to the respective microsomal proteins using appropriate biophysical techniques. The second phase will focus on the control of electron flux between the reductase and P-450 domains. The techniques used will complement those employed in phase one but will also include chemical modification, chemical cross-linking and site-directed mutagenesis to identify the residues and regions important in electron transfer and protein/protein recognition. The third phase, which will be conducted concurrently with the other two, is the determination of the three-dimensional structure of the holoenzyme and the individual domains. The results obtained will enable us to better understand the factors which control the oxidation of fatty acids, drugs, carcinogens, steroids, and xenobiotics by this class of P-450 enzymes