The lipoxygenase activity of myoglobin. Oxidation of linoleic acid by the ferryl oxygen rather than protein radical.

Linoleic (9(Z),12(Z)-octadecadienoic) acid is oxidized by sperm whale myoglobin and H2O2 to an 84:16 (9S):(9R) enantiomer mixture of 9-hydroperoxy-10(E),12(Z)-octadecadienoic acid. Neither the 9,10- nor 12,13-epoxide of linoleic acid, nor 9-hydroxy-10(E),12(Z)-octadecadienoic nor 13-hydroperoxy-9(Z),11(E)-octadeca-dienoic acids, is detectably formed. Incubations with [(11R)-2H]- and [(11S)-2H]linoleic acids indicate that the pro-R hydrogen is abstracted 76% of the time. An H64V mutant in which access to the heme crevice is increased oxidizes linoleic acid exclusively by abstraction of the pro-R hydrogen to give the (9S)-hydroperoxide. Spectroscopic studies show that the Kd value for binding of linoleic acid to myoglobin is similar to the Km value for its oxidation and indicate that linoleic acid reduces the ferryl species to the ferric state. The stereochemical results, supported by 18O-labeling studies, definitively rule out a significant role for singlet oxygen in the myoglobin-catalyzed, H2O2-dependent oxidation of linoleic acid. The myoglobin protein radical formed with H2O2 also plays no part in the reaction because the Km and Vmax values for the oxidation of linoleic acid are similar for native myoglobin and two mutants (K102Q/Y103F/Y146F/Y151F and H64V/K102Q/Y103F/Y146F/Y151F) with no tyrosine residues. Furthermore, the rate of formation of the 9-hydroperoxide is not changed if the protein radical is allowed to decay before linoleic acid is added. The results establish that linoleic acid is oxidized within the heme crevice by reaction with the ferryl oxygen rather than a protein radical. They indicate, furthermore, that hydrogen abstraction and oxygen addition occur in an antarafacial manner and suggest a specific model for binding of linoleic acid within the myoglobin active site.