NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein and requires derivatives of riboflavin, both FAD and FMN, for the transfer of electrons to the various cytochromes P450 (P450s) localized in the endoplasmic reticulum of the cells of most organs. The involvement of this P450-mediated electron transport system in the metabolism of both endogenous compounds derived from fatty acids and steroids (intracellular signals and hormones) and exogenous compounds (therapeutic drugs, environmental toxicants, and cancer-producing agents) reinforces its importance. This renewal application seeks to understand the mechanism by which CPR interacts with its oxidation-reduction partner(s), P450s. The planned studies will utilize the X-ray crystal structure recently determined by the Dr. Masters in collaboratio with Dr. J-J. P. Kim of the Medical College of Wisconsin to understand not onl the interactions of CPR with cytochromes P450 but structure-function aspects o the three isoforms of NOS, which contain a flavoprotein domain 58percent homologous to CPR and a heme domain with characteristics common to P450s.
Specific aims relating to the hypothesis that these enzyme systems utilize similar mechanisms to achieve diverse biological outcomes are as follows: I. Interactions of CPR with cytochromes P450: Identification of the residues involved in electron transfer from the flavoprotein to the heme protein utilizing site-directed and deletion mutants based upon CPR crystal structure and modeling of mammalian P450s based upon Bacillus megaterium P450 (BM-3).crystal structure. These mutants will be characterized by a variety of biophysical approaches, including optical and electron paramagnetic resonance spectroscopy, as well as determination of structures crystallographically. II. Mechanistic comparisons of CPR-P450s and NOSs: Utilizing reconstitution of activities in solution and recombinant DNA techniques to produce fusion proteins and chimeric constructs, these diverse enzyme systems will be studied for mechanistic comparison by both static and stopped-flow kinetic spectrophotometry. The results of these studies will enhance the understanding of flavoprotein-heme protein-containing systems, and enlighten the search for specific inhibitors, based on unique aspects of structure, possibly leading to more rational drug development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL030050-16A1
Application #
2605498
Study Section
Biochemistry Study Section (BIO)
Project Start
1982-06-01
Project End
2002-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
16
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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
Feng, Changjian; Roman, Linda J; Hazzard, James T et al. (2008) Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase. FEBS Lett 582:2768-72
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

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