Structural characterization of alkyl and peroxyl radicals in solutions of purple lipoxygenase

Soybean lipoxygenase isozyme 1 catalyzes the addition of dioxygen to fatty acid substrates that contain a 1,4-diene, generating allylic hydroperoxides. EPR spectra of purple enzyme solutions, formed by addition of saturating amounts of substrates or product to the enzyme, reveal the existence of fatty acid alkyl and peroxyl radicals that are bound to the enzyme and may be intermediates of the catalytic reaction [Nelson, M. J., Seitz, S. P., & Cowling, R. A. (1990) Biochemistry 29, 6897-6903]. We have analyzed the spectra of the radicals formed from the hydroperoxide products of four specifically deuterated linoleic acids and [per-2H]linoleic acid. The alkyl radical is an allyl radical, delocalized over C9 through C11 of linoleic acid. The data are consistent with delocalization of some of the spin over an unknown substituent at C12. The peroxyl radical is a 9-peroxyl derivative of linoleic acid. From the data we propose a novel mechanism for the lipoxygenase reaction: (1) oxidation of the 1,4-diene by the active-site Fe3+ to a delta 12-[9,10,11]-allyl radical; (2) activation of dioxygen at the Fe2+; (3) electrophilic attack by Fe(2+)-O2 on the 12-ene to form a 12,13-perepoxy-[9,10,11]-allyl radical; (4) opening of the perepoxide to the Fe(3+)-allylic hydroperoxide complex; (5) protonation to yield the 13-hydroperoxide. Addition of dioxygen to the allyl radical is proposed to form the 9-peroxyl, ultimately to yield the minor 9-hydroperoxide lipoxygenase product.