The contributions that this proposal seeks to make are to understand the structure of the native, activated and oxidatively inactivated lipoxygenases and to obtain thermodynamic parameters that can be applied to control lipoxygenase reactivity. 15-Lipoxygenases from soybeans and reticulocytes will be studied. An auxiliary goal is to broaden general knowledge of how the EPR spectra of iron enzymes can yield structural information The specific aims are to examine the following: (1)Are there two ferrous forms of lipoxygenase-l? Comparison of the resting ferrous protein and the ferrous form obtained by anaerobic reduction of the ferric form with substrate will be made. (2)The contribution of the iron center to stability of lipoxygenase-I will be examined using differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) to study thermal stability of lipoxygenase-1 in the absence and presence of reagents that interact with the iron site. (a)Experiments with native, ferrous lipoxygenase (b)Experiments with ferric and methionine oxidized lipoxygenase (c)Experiments with lipoxygenase in the presence of reagents that interact with the iron site (3)EPR and activity assays will be used to provide supporting information for collaborative work on x-ray structure analysis of lipoxygenases: effects of heavy atom reagents and salts will be examined and EPR studies of soybean lipoxygenase-I in the polycrystalline and crystalline states will be initiated. (4)The thermal stability of reticulocyte lipoxygenase will be examined using DSC, thermal loss of activity and EPR studies of the iron center. (5)The ability of reticulocyte lipoxygenase to oxidize unsaturated lipids in intact model lipid bilayers will be examined by oxygen uptake, calorimetry and chemical analysis. (6) Improved computer programs for analyzing metallo-EPR spectra will be developed..
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