title: EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.
abstract: The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.
authors.affiliation: Department of Chemistry, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606, USA.
authors.forename: Johanna
authors.initials: J
authors.lastname: Rapp
authors.forename: Shu
authors.initials: S
authors.lastname: Xu
authors.forename: Allan M
authors.initials: AM
authors.lastname: Sharp
authors.forename: Wendell P
authors.initials: WP
authors.lastname: Griffith
authors.forename: Yong-Wah
authors.initials: YW
authors.lastname: Kim
authors.forename: Max O
authors.initials: MO
authors.lastname: Funk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism*; D008054:Lipid Peroxides / Q000494:pharmacology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D034421:Sus scrofa; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D008054:Lipid Peroxides; D011994:Recombinant Proteins; D007501:Iron; D001092:Arachidonate 12-Lipoxygenase
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