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.
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