Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content

Abstract

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.

Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

“…We confirmed that the presence of the nitroxyl radical did not prevent the lipoxygenase reaction as follows: when 1.0 mM linoleate was incubated with 0.1 mM lipoxygenase in the presence or absence of CmDP (1.0 mM), there is no significant difference in the initial rate of hydroperoxide generation between the two systems (approximately 2.4 mM/sec). Remarkably, the nitroxyl radical scavenges the linoleate allyl radical on the ferrous lipoxygenase at lower oxygen content, resulting in the release of the inactive ferrous lipoxygenase (13). It is generally known that lipid hydroperoxides play a key role in the conversion of ferrous lipoxygenase into active ferric lipoxygenase, generating lipid alkoxyl radicals (14-16), which easily turn into epoxyallyl radicals through intramolecular rearrangement.…”

“…In both cases, lipid allyl radical-CmDP adducts and lipid epoxyallyl radical-CmDP adducts were detected. The scavenging of lipid allyl radicals on the ferrous lipoxygenase by the nitroxyl radical promotes the one-electron redox cycle reaction (13).…”

“…This fact indicates that the mobility of the framework of the linolenate 9-peroxyl radical is not necessarily retarded by fixation in the active site of lipoxygenase. In our recent study, we found that the binding affinity of the lipid allyl radical to the active site on lipoxygenase at lower oxygen content is not so tight that the bulky nitroxyl radical can scavenge the lipid allyl radical (13). In a similar manner, the linolenate 9-peroxyl radical is thought to be flexible in the active site on lipoxygenase at lower oxygen content, allowing the intramolecular rearrangement of the linolenate 9-peroxyl radical.…”

Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid

“…As shown in Fig. 1, coexisting nitroxyl radical (CmDP) scavenges the FA allyl radical on the lipoxygenase, producing FA allyl radicalnitroxyl radical adducts and ferrous lipoxygenase (11). Subsequently, the ferrous lipoxygenase should be reoxidized to ferric one by cycling hydroperoxides through a pseudoperoxidase reaction, generating a FA alkoxyl radical, which may be intramolecularly rearranged by the addition of a double bond to form the FA epoxyallyl radical (carbon-centered radical) (16,17).…”

“…In our previous reports (11,12), we established a novel method for converting ferric lipoxygenase into its ferrous form. When soybean ferric lipoxygenase was incubated with PUFAs in the presence of nitroxyl radical, which selectively traps carbon-centered radicals, FA allyl radical on the ferrous lipoxygenase (13-15) was trapped by nitroxyl radical, generating ferrous lipoxygenase.…”