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 the fungus Gaumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[H-2]- and (11S)-[H-2]linoleic acids with Mn-LO led to the formation of hydroperoxides that largely retained and lost, respectively, the deuterium label. Conversion of the (11S)-deuteriolinoleic acid was accompanied by a primary isotope effect, which manifested itself in a strongly reduced rate of formation of hydroperoxides and in a time dependent accumulation of deuterium in the unconverted substrate. 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 hydroperoxide 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 retained 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)-HPOD suffered a time-dependent loss of O-18 when exposed to Mn-LO, whereas (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 conversion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of this intermediate by dioxygen attack at C-13 produced (13R)-HPOD, whereas attack at C-ll regenerated (11S)-HPOD, The hydroperoxide rearrangement occurred by oxygen rebound, although, as demonstrated by the O-18 experiments, the oxygen molecule released from (11S)-HPOD exchanged with surrounding molecular oxygen prior to its reincorporation.