Factors affecting the line-shape of the EPR signal of high-spin Fe(III) in soybean lipoxygenase-1

doi:10.1016/0167-4838(82)90435-6

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 708, Issue 3, 19 November 1982, Pages 259-265

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(82)90435-6 Get rights and content

Abstract

The yellow form of soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12), obtained upon addition of one molar equivalent of $13- l_{s} - hydroperoxy -9-cis,11-trans- octadecadienoic$ acid (13-l-HPOD) to the native enzyme, shows a complex EPR signal around g 6 which results from contributions of different high-spin Fe(III) species with rhombic or axial symmetry. The signal cannot be attributed to different enzyme-product complexes because removal of the products or variation of the concentration of the products in the enzyme solution does not lead to an EPR spectrum characteristic for one particular species. Upon varying the pH of the enzyme solution from 7 to 11 changes in the line-shape are observed, but no distinct spectrum of either a rhombic or an axial form could be observed. The relative amounts of the different species visible in the signal around g 6 are strongly affected by cyanide, primary alcohols or 13-l-hydroxyoctadecadienoic acid (reduced 13-l- HPOD). The presence of either of these substances causes a shift to an axial type of spectrum, t-Butanol and sodium dodecyl sulfate induce a shift towards a more rhombic line-shape. A shift to an axial type of spectrum is observed after storage of the enzyme in the yellow form at 4°C. Storage of the native form at 4°C also leads to changes which become apparent after oxidation in a similar axial type of spectrum. Reversion to the original, more rhombic, spectrum is possible by ammonium sulfate precipitation. It is concluded that the species giving rise to the EPR signal around g 6 are enzyme species differing only in the structure of the environment of the iron atom.

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Citation Excerpt :
The heterogeneity of the g6 signal of soybean lipoxygenase-1 was sensitive to the composition of the sample. For example, distinctively more homogeneous g6 signals were obtained when low molecular weight alcohols were present [35]. Human and potato 5-lipoxygenase both produced heterogeneous g6 EPR signals as well as signals at g4.3 upon oxidation [33,34].
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 25 K 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.
Inhibition of lipoxygenase by soy isoflavones: Evidence of isoflavones as redox inhibitors
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Soy LOX–1 contains a nonheme ferrous ion that is oxidized by hydroperoxides to yield the catalytically active ferric enzyme [32]. The ferric enzyme can be distinguished from the ferrous form by its unique CD and EPR features—appearance of a positive band at 425 nm [30] and signal at g = 6.1 [31], respectively. Antioxidants inhibit lipoxygenase activity by reducing the enzyme bound fatty acid radical intermediates—L and LOO[32,33].
Hydroperoxides, the products of lipoxygenase mediated pathways, play a major role in the manifestation of chronic inflammatory diseases. Soy isoflavones act as antioxidants due to their ability to scavenge free radicals. Isoflavones inhibit the activity of soy lipoxygenase-1 and 5-lipoxygenase, from human polymorph nuclear lymphocyte in a concentration dependent manner. Spectroscopic and enzyme kinetic measurements have helped to understand the nature and mechanism of inhibition. Genistein is the most effective inhibitor of soy lipoxygenase 1 and 5-lipoxygenase with IC₅₀ values of 107 and 125 μM, respectively. Genistein and daidzein are noncompetitive inhibitors of soy lipoxygenase 1 with inhibition constants, K_i, of 60 and 80 μM, respectively. Electron paramagnetic resonance and spectroscopic studies confirm that isoflavones reduce active state iron to ferrous state and prevent the activation of the resting enzyme. A model for the inhibition of lipoxygenase by isoflavones is suggested.
Inhibition of soybean lipoxygenase-1 by n-alcohols and n-alkylthiols
1992, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
A series of n-alcohols and n-alkylthiols with carbon chains from 2 to 12 were examined for the inhibition of soybean lipoxygenase-1 (L-1). The alcohol produces a competitive inhibition, the extent of which increases with an increase in the carbon number of alkyl chain up to 8. Whereas the inhibition of the alkylthiol is noncompetitive, the extent of which is almost independent from the carbon number. From the behavior of pK_i dependence on the carbon number of the alcohol, the decyl group appears to be optimum to bind to L-1. The thermodynamic analysis for the inhibition based upon van 't Hoff equation indicates positive enthalpy and entropy changes for the binding of the alcohol to the enzyme and negative enthalpy and positive to negative entropy changes for that of the alkylthiol. These observations suggest that the alcohol inhibits L-1 by binding of the hydrophobic alkyl tail to the catalytic site of the enzyme by a hydrophobic interaction. The alkylthiol inhibits by binding of the nucleophilic sulfhydryl head to a polarizable region of the enzyme and the alkyl tail to a hydrophobic region of the enzyme free from the steric hindrance as an anchor.
Monomeric spin sextet paramagnets
1992, Studies in Inorganic Chemistry
Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. I. NO binds to the acceptor-side non-heme iron
1990, BBA - Bioenergetics
NO is a good electrophile and EPR spin probe, carrying an unpaired electron ( $S = 12$ ). Exposure of Photosystem II reaction centers to NO results in the appearance of an EPR signal at g = 4. Dark titration with NO shows a K_d ≈ 30 μM in spinach chloroplasts and 250 μM in BBY preparations. Successive cycles of illumination at 200 K, followed by incubation at 245 K, results in binary oscillations of the amplitude of the g = 4 signal in spinach chloroplasts. This signal is small in states Q⁻_AQ_B, Q⁻_AQ⁻_B, but large in states Q_AQ_B and Q_AQ_BH₂ of the PS II acceptor side. NO slows electron transfer between Q_A and Q_B (Diner, B.A and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141–149) and modifies the Mössbauer spectrum of the non-heme Fe(II). These results strongly support the assignment of the g = 4 EPR signal to an acceptor side Fe(II)-NO adduct in an $S = 32$ state. Exchange coupling of this species with the $S = 12$ semiquinones results in an integral spin system, not detectable in X-band EPR. The g = 4 spectrum can be described by a spin hamiltonian. The rhombicity parameter $E D$ is in all cases small (⩽ 0.015), implying a near axial environment. NO can donate electrons to PS II. Charge recombination, following a light flash in the presence of DCMU, is blocked by NO (Km ≈ 30 μM, 25°C). NO also reacts reversibly in the dark with D⁺ (an oxidized secondary donor), resulting in the disappearance of Signal II_dark with a K_d of 3 μM. Pumping off of NO in the dark results in full recovery of the signal.
Lipoxygenase isoenzymes: A spectroscopic and structural characterization of soybean seed enzymes
1989, Archives of Biochemistry and Biophysics
Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

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Factors affecting the line-shape of the EPR signal of high-spin Fe(III) in soybean lipoxygenase-1

Abstract

Biochim. Biophys. Acta

FEBS Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

FEBS Lett.

Biochim. Biophys. Acta

Biochim. Biophys. Acta