EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.

Rapp J, Xu S, Sharp AM, Griffith WP, Kim YW, Funk MO
Arch Biochem Biophys. 2009 490 (1): 50-6

PMID: 19683507 · PMCID: PMC2778285 · DOI:10.1016/j.abb.2009.08.004

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.

MeSH Terms (16)

Amino Acid Sequence Animals Arachidonate 12-Lipoxygenase Electron Spin Resonance Spectroscopy Escherichia coli Hydrogen-Ion Concentration Iron Leukocytes Lipid Peroxides Molecular Weight Oxidation-Reduction Recombinant Proteins Spectrometry, Mass, Electrospray Ionization Sus scrofa Temperature Transformation, Genetic

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