MICROSOMAL ELECTRON TRANSPORT IN LIVER AND HEART

  • Masters, Bettie S (PI)

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date6/1/823/31/07

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $218,250.00
  • National Institutes of Health
  • National Institutes of Health: $206,985.00
  • National Institutes of Health
  • National Institutes of Health: $166,979.00
  • National Institutes of Health: $124,902.00
  • National Institutes of Health: $244,954.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $205,104.00
  • National Institutes of Health: $214,955.00
  • National Institutes of Health: $218,563.00
  • National Institutes of Health: $219,000.00
  • National Institutes of Health: $190,951.00
  • National Institutes of Health
  • National Institutes of Health: $195,037.00
  • National Institutes of Health

ASJC

  • Medicine(all)

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