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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37HL030050-06A1
Application #
3485975
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1982-06-01
Project End
1993-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Kranendonk, Michel; Marohnic, Christopher C; Panda, Satya P et al. (2008) Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase. Arch Biochem Biophys 475:93-9
Erdal, Erik P; Martasek, Pavel; Roman, Linda J et al. (2007) Hydroxyethylene isosteres of selective neuronal nitric oxide synthase inhibitors. Bioorg Med Chem 15:6096-108
Seo, Jiwon; Martasek, Pavel; Roman, Linda J et al. (2007) Selective L-nitroargininylaminopyrrolidine and L-nitroargininylaminopiperidine neuronal nitric oxide synthase inhibitors. Bioorg Med Chem 15:1928-38
Gao, Ying Tong; Roman, Linda J; Martasek, Pavel et al. (2007) Oxygen metabolism by endothelial nitric-oxide synthase. J Biol Chem 282:28557-65
Gao, Ying Tong; Panda, Satya Prakash; Roman, Linda J et al. (2007) Oxygen metabolism by neuronal nitric-oxide synthase. J Biol Chem 282:7921-9
Panda, Satya Prakash; Gao, Ying Tong; Roman, Linda J et al. (2006) The role of a conserved serine residue within hydrogen bonding distance of FAD in redox properties and the modulation of catalysis by Ca2+/calmodulin of constitutive nitric-oxide synthases. J Biol Chem 281:34246-57
Mbadugha, Bessie N A; Seo, Jiwon; Ji, Haitao et al. (2006) Hydroxyl-terminated peptidomimetic inhibitors of neuronal nitric oxide synthase. Bioorg Med Chem 14:3681-90
Marohnic, Christopher C; Panda, Satya P; Martasek, Pavel et al. (2006) Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase. J Biol Chem 281:35975-82
Whitsett, Jennifer; Martasek, Pavel; Zhao, Hongtao et al. (2006) Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution. Free Radic Biol Med 40:2056-68
Ji, Haitao; Gomez-Vidal, Jose A; Martasek, Pavel et al. (2006) Conformationally restricted dipeptide amides as potent and selective neuronal nitric oxide synthase inhibitors. J Med Chem 49:6254-63

Showing the most recent 10 out of 56 publications