Understanding the radical mechanism of lipoxygenases using 31P NMR spin trapping

https://doi.org/10.1016/j.bmc.2011.02.046Get rights and content

Abstract

In this paper, we use our quantitative 31P NMR spin trapping methods, already developed for simple oxygen- and carbon-centered radicals, to understand the radical intermediates generated by enzymatic systems and more specifically lipoxygenases. Our methodology rests on the fact that free radicals react with the nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using 31P NMR in the presence of a phosphorus containing internal standard. This system was thus applied to better understand the mechanism of enzymatic oxidation of linoleic acid by soybean lipoxygenases-1 (LOX). The total amount of radicals trapped by DIPPMPO was detected by 31P NMR at different experimental conditions. In particular the effect of dioxygen concentration on the amount of radicals being trapped was studied. At low dioxygen concentration, a huge increase of radicals trapped was observed with respect to the amount of radicals being trapped at normal dioxygen concentrations.

Introduction

Lipoxygenases (LOXs) form a heterogeneous family of lipid peroxidizing enzymes that catalyze dioxygenation of polyunsaturated fatty acids (PUFA) to their corresponding hydroperoxy derivatives.1 In animal tissues, the most common substrate encountered is the arachidonic acid (C20:4), which is dioxygenated by lipoxygenases into precursors of products involved into inflammatory processes,2 cell membranes maturation,3 cancer metastases,4, 5 atherogenesis6, 7 and osteoporosis.8 The role of plant lipoxygenases, whose main substrates are linoleic (C18:2) and linolenic acids (C18:3), is not very fully elucidated, although they are implied in processes such as senescence or plant response to wounding.9

A single non-heme iron is present in each enzyme and it exists in two oxidation states: Fe(II) and Fe(III).10 According to the current working mechanism,10, 11 the native enzyme is inactive in the Fe(II) form. When treated with an equimolar amount of product, the iron is oxidized to the Fe(III) form, resulting in an active enzyme. The ferric form can then catalyze the abstraction of a hydrogen from the bis-allylic carbon atom of the substrate from 11 position of linoleic acid, in a stereospecific manner, yielding a pentadienyl radical complexed with the ferrous enzyme. Bimolecular oxygen is then inserted to the pentadienyl radical, through a channel in the lipoxygenase, which leads to the formation of the hydroperoxide product and the reoxidation of the cofactor to the ferric form (Scheme 1).

Although the LOX-reaction involves the formation of radical intermediates it may not be considered an effective source of free radicals as most of the intermediates remain enzyme bound. However, under certain conditions a considerable portion of radicals may escape the active site leaving the enzyme in the inactive form Fe(II).12, 13 These observations were based on the change in LOXs regiospecificity at different dioxygen concentrations. In fact, under most conditions, soybean lipoxygenase-1 is highly specific for the position at which the dioxygen is inserted (position 13). However, this specificity can be greatly influenced by the dioxygen concentration.13

In order to obtain direct evidence of the increase in the radical escaping mechanism from the enzyme active site at different dioxygen concentrations, a spin trap technique could be applied. Using the 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO) as spin trap, the presence of phosphorus allows for the use of phosphorus nuclear magnetic resonance (31P NMR) spectrometry to investigate the detailed chemistry of the radical reaction. 31P NMR could be exploited to perform quantitative analyses, in the presence of a suitable internal standard.14 Early qualitative work showed that the chemical shift of the 31P atom depends on the nature of the adducts.15, 16

In the present study we have attempted to determine and explain the influence of dioxygen concentration on soybean lipoxygenase-1 radical generation. The dioxygen concentration in a reaction solution is a function of two parameters: the initial dioxygen concentration and the rate of consumption. As such we have systematically varied the initial and subsequent oxygenation conditions (argon head-space, air bubbling) and have been able to determine and quantify the radical intermediates involved under the different conditions.

Section snippets

Chemicals

Soybean lipoxygenase-1 (type I-B, activity 150,000 U/mg), linoleic acid and all chemicals were purchased from Sigma (St. Louis, MO, USA) and used as received. DIPPMPO was synthesized as describe below. All reagents and buffers were prepared using Millipore MilliQ deionized water (ρ = 18  cm).

Synthesis of DIPPMPO

DIPPMPO (5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide) was synthesized according to the literature.17 The spin trap was stored under argon at −78 °C.

Linoleic acid oxidation by lipoxygenase

The linoleic acid oxidation was carried out at room

Results and discussion

Soybean lipoxygenase-1 shares good sequence identity with mammalian lipoxygenases, and its X-rays crystal structure reveals similar details with that of the rabbit 12/15-lipoxygenase. For this reason, soybean lipoxygenase-1 has been used in place of the mammalian enzyme.18, 19, 20 Qian et al.21, 22, 23 examined the free radical generation in the reaction between soybean lipoxygenase-1 and linoleic acid by using nitrone spin trapping including DMPO (5,5′-dimethyl-1-pyrroline-N-oxide) and POBN

References and notes (29)

  • H. Kuhn et al.

    FEBS Lett.

    (1999)
  • T. Schewe

    Trends Biochem. Sci.

    (1991)
  • M.K. Cathcart et al.

    Free Rad. Biol. Med.

    (2000)
  • H.W. Gardner

    Biochim. Biophys. Acta

    (1991)
  • J. De Groot et al.

    Biochim. Biophys. Acta

    (1975)
  • H. Berry et al.

    J. Biol. Chem.

    (1998)
  • D.S. Argyropoulos et al.

    Bioorg. Med. Chem.

    (2006)
  • L. Zoia et al.

    J. Phys. Org. Chem.

    (2010)
  • S.Y. Qian et al.

    Free Radical Biol. Med.

    (2002)
  • S.Y. Qian et al.

    Free Radical Biol. Med.

    (2003)
  • S.Y. Qian et al.

    Free Radical Biol. Med.

    (2003)
  • A. Reis et al.

    J. Am. Soc. Mass. Spectrom.

    (2003)
  • B. Samuelsson

    Science

    (1987)
  • K.V. Honn et al.

    Cancer Metastasis Rev.

    (1994)
  • Cited by (14)

    • Structural and functional evaluation mammalian and plant lipoxygenases upon association with nanodics as membrane mimetics

      2022, Biophysical Chemistry
      Citation Excerpt :

      Polymer based nanodiscs, although smaller in size and amenable to tuning based on polymer composition and pH, nonetheless were also shown to be susceptible to aggregation in the presence of divalent cations [38,39]. Although their Ca2+ dependent membrane association [40] and enzymatic reaction [41] are similar, mammalian ALOX15, ALOX15B and soybean LOX-1 have only 25% similarity in their amino acid sequence. Structural alignment between the three proteins shows that mammalian LOXs have shorter loops which make them more compact while LOX-1 has an additional 35 amino acid segment that makes it bigger (Fig. 3A-C).

    • Lipoxygenase-mediated peroxidation of model plant extractives

      2017, Industrial Crops and Products
      Citation Excerpt :

      Moreover, the produced complex is extremely unstable and can easily react with the dioxygen to form a new proxy radical complex (LOX-LOO·). The group that is produced by the reaction can be stabilized during the LOX catalytic cycle via an intra-complex electron transfer process, which reduces the radical to its anion (LOO−) (Zoia et al., 2011). As shown in Fig. 2a, this is the intermediate product for the formation of the hydroperoxy group; however it is not clear what portion of the LOO− groups forms the HOO groups and what portion remains unchanged.

    • Antioxidant and lipoxygenase activities of polyphenol extracts from oat brans treated with polysaccharide degrading enzymes

      2017, Heliyon
      Citation Excerpt :

      In addition to free radicals, enzymes such lipoxygenases (LOX), which oxidize unsaturated fatty acids (e.g. arachidonic acid and linoleic acid) also contributed to the increase risks of chronic diseases, specifically inflammation. LOX action generates hydroperoxides that may undergo further reactions to produce volatile carbonyls [7, 8]. Molecules that scavenge free radicals or inhibit the activity of LOX may then serve as modulators of the inflammatory process.

    • Identification and quantification of radical species by <sup>31</sup>P NMR-based spin trapping - A case study: NH<inf>4</inf>OH/H<inf>2</inf>O<inf>2</inf>-based hair bleaching

      2015, Microchemical Journal
      Citation Excerpt :

      Overall, however, a possible drawback of this technique could be the reduced sensitivity of NMR compared to that of EPR. This is partly overcome by the acquisition of more NMR signals with time [18]. Due to the presence of the phosphorous atom in the DIPPMPO spin trap, 31P NMR spectroscopy can be conveniently used for both qualitative and quantitative analyses, in the presence of a suitable internal standard, benefitting from the fact that different spin trap adducts show different chemical shifts of the 31P atom, depending on the nature of the adduct forming radicals.

    • A method for simultaneous quantification of phospholipid species by routine <sup>31</sup>P NMR

      2012, Journal of Pharmaceutical and Biomedical Analysis
      Citation Excerpt :

      However, in NMR spectroscopy, direct quantitative analysis is possible, even though this is not common on a routine basis. Therefore, internal standards are frequently used for quantitative NMR techniques [12–18]. Marinier et al. [13] report the use of 0.005% (v/v) trimethylphosphate (TMP) added to each sample.

    View all citing articles on Scopus
    View full text