MANGANESE LIPOXYGENASE - DISCOVERY OF A BIS-ALLYLIC HYDROPEROXIDE AS PRODUCT AND INTERMEDIATE IN A LIPOXYGENASE REACTION

Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from t he fungus Gaumannomyces graminis. The product consisted of (13R)-hydro peroxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperox ide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Inc ubation of (11R)-[H-2]- and (11S)-[H-2]linoleic acids with Mn-LO led t o the formation of hydroperoxides that largely retained and lost, resp ectively, the deuterium label. Conversion of the (11S)-deuteriolinolei c acid was accompanied by a primary isotope effect, which manifested i tself in a strongly reduced rate of formation of hydroperoxides and in a time dependent accumulation of deuterium in the unconverted substra te. These experiments indicated that the initial step catalyzed by Mn- LO consisted of abstraction of the pro-S hydrogen of linoleic acid to produce a linoleoyl radical. (11S)-HPOD was converted into (13R)-HPOD upon incubation with Mn-LO. The mechanism of this enzyme-catalyzed hyd roperoxide rearrangement was studied in experiments carried out with O -18(2) gas or O-18(2)-labeled hydroperoxides, Incubation of [11-O-18(2 )](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD, which reta ined 39-44% of the O-18 label, whereas (11S)-HPOD incubated with Mn-LO under O-18(2) produced (13R)-HPOD, which had incorporated 57% of O-18 . Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that [11-O-18(2)](11S)-H POD suffered a time-dependent loss of O-18 when exposed to Mn-LO, wher eas (11S)-HPOD incorporated O-18 when incubated with Mn-LO under O-18( 2). On the basis of these experiments, it was proposed that the conver sion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of th is intermediate by dioxygen attack at C-13 produced (13R)-HPOD, wherea s attack at C-ll regenerated (11S)-HPOD, The hydroperoxide rearrangeme nt occurred by oxygen rebound, although, as demonstrated by the O-18 e xperiments, the oxygen molecule released from (11S)-HPOD exchanged wit h surrounding molecular oxygen prior to its reincorporation.