) Human mitochondrial superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion, 2O2- + 2H+ - 02 + H202, and is important as a main line of defense against oxidative damage in normal metabolism and in a number of disease states. This catalysis requires proton transfers ultimately from solution to the active site to release product hydrogen peroxide, and is accompanied by a prominent product inhibition. The unifying goals of this proposal are to elucidate the function of active-site residues in the catalytic mechanism and in the product inhibition emphasizing the role of proton transfer and the function of an envelope of hydrophobic residues that surround the metal. We plan structure-function studies using site-specific mutagenesis to alter residues near the active site. Pulse radiolysis and scanning stopped-flow spectrophotometry will be used to evaluate changes in catalysis and x-ray crystallography to direct structural changes. We will also investigate the function of these residues in the fine tuning of the redox potential. Potentiometric titrations will be performed to determine the reduction midpoint potentials of mutants and to discern the influence of active site residues, information which will also be related to the rates of catalysis and extent of product inhibition. Catalysis by MnSOD will be activated by enhancing the rate of protein transfer to the active site using exogenous proton donors and intramolecular shuttle groups. Marcus rate theory will be applied to these data to determine the intrinsic kinetic barrier and thermodynamic components of proton transfer in MnSOD, data that will be compared with proton transfers in other systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054903-05A1
Application #
6326884
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Jones, Warren
Project Start
1996-09-01
Project End
2005-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
5
Fiscal Year
2001
Total Cost
$253,706
Indirect Cost
Name
University of Florida
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Perry, J Jefferson P; Hearn, Amy S; Cabelli, Diane E et al. (2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis. Biochemistry 48:3417-24
Abreu, Isabel A; Hearn, Amy; An, Haiqain et al. (2008) The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations. Biochemistry 47:2350-6
Quint, Patrick S; Domsic, John F; Cabelli, Diane E et al. (2008) Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase. Biochemistry 47:4621-8
Emmler, Th; Ayala, I; Silverman, D et al. (2008) Combined NMR and computational study for azide binding to human manganese superoxide dismutase. Solid State Nucl Magn Reson 34:6-13
Zheng, Jiayin; Domsic, John F; Cabelli, Diane et al. (2007) Structural and kinetic study of differences between human and Escherichia coli manganese superoxide dismutases. Biochemistry 46:14830-7
Quint, Patrick; Reutzel, Robbie; Mikulski, Rose et al. (2006) Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation. Free Radic Biol Med 40:453-8
Chockalingam, Karuppiah; Luba, James; Nick, Harry S et al. (2006) Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition. FEBS J 273:4853-61
Quint, Patrick; Ayala, Idelisa; Busby, Scott A et al. (2006) Structural mobility in human manganese superoxide dismutase. Biochemistry 45:8209-15
Tu, Chingkuang; Quint, Patrick; Silverman, David N (2005) Exchange of (18)O in the reaction of peroxynitrite with CO(2). Free Radic Biol Med 38:93-7
Ayala, Idelisa; Perry, J Jefferson P; Szczepanski, Jan et al. (2005) Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine. Biophys J 89:4171-9

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