MICROSOMAL ELECTRON TRANSPORT IN LIVER AND HEART

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Because NADPH-cytochrome P-450 reductase exists in every tissue in which the cytochrome P-450-mediated hydroxylations of both endogenous (steroids, fatty acids, and prostaglandins) and exogenous (therapeutic drugs, environmental toxicants and carcinogens) occur, it is important to understand its mode of action. This proposal is aimed at understanding the structure- function relationships of the liver microsomal flavoprotein, NADPH- cytochrome P-450 reductase, which contains both FAD and FMN as prosthetic groups-a unique among mammalian flavoenzymes. In interacting with its physiological electron acceptor, cytochrome(s) P-450, this flavoprotein exercises a mechanism which allows the insertion of 2 electrons sequentially into the substrate-bound- cytochrome P-450 reduced 02 complex. This process requires a unique conformation with distinct structural domains for the binding of each of the prosthetic flavins and the capability of generating the appropriate oxidation-reduction states to interact with specific redox states of cytochrome P-450 during catalytic turnover. Due to the fact that no single technique can address the various aspects of this interesting and vital flavoprotein, we plan to examine its structure and function at the molecular level by a variety of biophysical methods. We will perform: 1) 31P NMR studies on the native pig and rat reductases and on enzymes substituted with phosphorothioate analogs of both FMN and FAD and on site-directed mutagenesis products of rat liver reductase to determine effects on FMN-, and NADPH-binding domains as detected by line broadening and/or chemical shifts; 2) complementary and supplementary studies with laser resonance Raman spectroscopy on aliquots of the NMR samples of reductase, on enzyme with FMN substituted with 13C and 15N in the isoalloxazine ring, and on the mutant reductases to probe the environment of the flavins (hydrogen bonding effects); 3) studies on the crystallization of both intact and proteolytically cleaved reductase for X-ray crystallography studies; and 4) determination of the nature of reductase-bound phosphorus (bound phospholipid?) and its functional role. This combination of techniques will permit a comprehensive and, hopefully, conclusive study of the structure-function properties of this unique mammalian flavoprotein.