A PROTEIN RADICAL AND FERRYL INTERMEDIATES ARE GENERATED BY LINOLEATEDIOL SYNTHASE, A FERRIC HEMEPROTEIN WITH DIOXYGENASE AND HYDROPEROXIDE ISOMERASE ACTIVITIES

Linoleate diol synthase (LDS) was isolated as a hemeprotein from the f ungus Gaeumannomyces graminis, LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertio n of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydrop eroxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mai nly high spin. Oxygen consumption during catalysis started after a sho rt time lag which was reduced by 8-HPODE, Catalysis declined due to su icide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 de grees C showed a rapid decrease in light absorption at 406 nm within 3 5 ms with a first order rate constant of 90-120 s(-1). Light absorptio n at 406 nm then increased at a rate of similar to 4 s(-1), whereas th e absorption at 421 nm increased after a lag time of similar to 5 ms a t a rate of similar to 70 s(-1). EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched aft er incubation on ice for 3 s (major hyperfine splitting 2.3 millitesla ; g = 2.005), indicating a protein radical. The relaxation properties of the protein radical suggested interaction with a metal center. 8-HP ODE generated about twice as much radical as linoleic acid, and the 8- HPODE-induced radical appeared to be stable. Our results suggest that LDS may form, in analogy with prostaglandin Ii synthases, ferryl inter mediates and a protein radical during catalysis.