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Journal of Lipid Research, Vol. 49, 420-428, February 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Ernst H. Oliw¹

Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden

Published, JLR Papers in Press, November 17, 2007.

¹ To whom correspondence should be addressed. e-mail: ernst.oliw{at}farmbio.uu.se

Manganese lipoxygenase (Mn-LOX) catalyzes the rearrangement of bis-allylic S-hydroperoxides to allylic R-hydroperoxides, but little is known about the reaction mechanism. 1-Linoleoyl-lysoglycerophosphatidylcholine was oxidized in analogy with 18:2n-6 at the bis-allylic carbon with rearrangement to C-13 at the end of lipoxygenation, suggesting a "tail-first" model. The rearrangement of bis-allylic hydroperoxides was influenced by double bond configuration and the chain length of fatty acids. The Gly316Ala mutant changed the position of lipoxygenation toward the carboxyl group of 20:2n-6 and 20:3n-3 and prevented the bis-allylic hydroperoxide of 20:3n-3 but not 20:2n-6 to interact with the catalytic metal. The oxidized form, Mn^III-LOX, likely accepts an electron from the bis-allylic hydroperoxide anion with the formation of the peroxyl radical, but rearrangement of 11-hydroperoxyoctadecatrienoic acid by Mn-LOX was not reduced in D₂O (pD 7.5), and aqueous Fe³⁺ did not transfer 11S-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid to allylic hydroperoxides. Mutants in the vicinity of the catalytic metal, Asn466Leu and Ser469Ala, had little influence on bis-allylic hydroperoxide rearrangement. In conclusion, Mn-LOX transforms bis-allylic hydroperoxides to allylic by a reaction likely based on the positioning of the hydroperoxide close to Mn³⁺ and electron transfer to the metal, with the formation of a bis-allylic peroxyl radical, β-fragmentation, and oxygenation under steric control by the protein.

Supplementary key words electron transfer • 1-linoleoyl-lysoglycerophosphatidylcholine • mass spectrometry • metalloenzymes • peroxyl radicals • R-lipoxygenase • solvent deuterium isotope effect • site-directed mutagenesis

Abbreviations: CP, chiral phase; DiHETrE, dihydroxyeicosatrienoic acid; GPC, glycerophosphatidylcholine; HEDE, hydroxyeicosadienoic acid; HODE, hydroxyoctadecadienoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HPETrE, hydroperoxyeicosatrienoic acid; HPODE, hydroperoxyoctadecadienoic acid; HPOTrE, hydroperoxyoctadecatrienoic acid; Mn-LOX, manganese lipoxygenase; NP, normal phase; RP, reverse phase; sLO-1, soybean lipoxygenase; UV, ultraviolet

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