STRUCTURAL CHARACTERIZATION OF ALKYL AND PEROXYL RADICALS IN SOLUTIONS OF PURPLE LIPOXYGENASE

Soybean lipoxygenase isozyme 1 catalyzes the addition of dioxygen to f atty acid substrates that contain a 1,4-diene, generating allylic hydr operoxides. 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 Nels on, M. J., Seitz, S. P., & Cowling, R. A. (1990) Biochemistry 29, 6897 -6903. We have analyzed the spectra of the radicals formed from the h ydroperoxide products of four specifically deuterated linoleic acids a nd per-H-2linoleic acid. The alkyl radical is an allyl radical, delo calized 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 reacti on: (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 Fe2+-O-2 on the 12-ene to form a 12,13-per epoxy-9,10,11-allyl radical; (4) opening of the perepoxide to the Fe 3+-allylic hydroperoxide complex; (5) protonation to yield the 13-hydr operoxide. Addition of dioxygen to the allyl radical is proposed to fo rm the 9-peroxyl, ultimately to yield the minor 9-hydroperoxide lipoxy genase product.