The overall goal of the studies proposed is to develop detailed understanding of how the enzymes interacting with hydroperoxides of polyunsaturated fatty acids (PUFA) cause electron redistribution in their free radical intermediates. In eukaryotic cells, hydroperoxides of polyunsaturated fatty acids (PUFA) are formed and rapidly converted to signaling molecules in dynamic complexes of several enzymes. The balance of products resulting from separate PUFA oxidation pathways is a subject critical to current pharmaceutical treatment of inflammatory and cardiovascular disorders. This proposal focuses on the mechanisms of electron rearrangements in two representative enzymes forming, and acting on, PUFA hydroperoxides. The specific enzymes chosen for study include manganese lipoxygenases (MnLO and mutants of iron lipoxygenase), that form PUFA hydroperoxides and an animal allene oxide synthase (AOS), that converts PUFA hydroperoxides to downstream signals. Each of the enzymes is chosen because of novel catalytic features.
The specific aims are: (1) To examine, by high frequency electron paramagnetic resonance (hfEPR), activation of MnLO by two of its products, 11S- and 13R-hydroperoxides of linoleic acid. (2) To compare reduction potentials of manganese and iron in lipoxygenases using hfEPR. (3) To examine hfEPR spectroscopy and reduction potential of iron lipoxygenase mutated to include sequences that specify manganese binding. (4) To characterize functions and oxidation kinetics of AOS mutated at native tyrosine sites. (5) To obtain very high frequency EPR (vhfEPR) data on the microenvironment of the AOS tyrosine radical. The sensitivity of high frequency EPR (94 GHz), and simulation of EPR spectra, will facilitate many of these studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM065268-01A1
Application #
6616508
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Cassatt, James
Project Start
2003-04-10
Project End
2007-03-31
Budget Start
2003-04-10
Budget End
2004-03-31
Support Year
1
Fiscal Year
2003
Total Cost
$205,487
Indirect Cost
Name
Florida State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
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Bradshaw, Miles D; Gaffney, Betty J (2014) Fluctuations of an exposed ?-helix involved in lipoxygenase substrate recognition. Biochemistry 53:5102-10
Garreta, Albert; Val-Moraes, Silvana P; García-Fernández, Queralt et al. (2013) Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. FASEB J 27:4811-21
Gaffney, Betty J; Bradshaw, Miles D; Frausto, Stephen D et al. (2012) Locating a lipid at the portal to the lipoxygenase active site. Biophys J 103:2134-44
Gaffney, Betty J (2009) EPR of Mononuclear Non-Heme Iron Proteins. Biol Magn Reson 28:233-268
Wu, Fayi; Gaffney, Betty J (2006) Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1. Biochemistry 45:12510-8
Youn, Buhyun; Sellhorn, George E; Mirchel, Ryan J et al. (2006) Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3. Proteins 65:1008-20
Coffa, Gianguido; Imber, Ann N; Maguire, Brendan C et al. (2005) On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant. J Biol Chem 280:38756-66
Agarwalla, Sanjay; Stroud, Robert M; Gaffney, Betty J (2004) Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies. J Biol Chem 279:34123-9
Wu, Fayi; Katsir, Leron J; Seavy, Margaret et al. (2003) Role of radical formation at tyrosine 193 in the allene oxide synthase domain of a lipoxygenase-AOS fusion protein from coral. Biochemistry 42:6871-80