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J Biol Chem, Vol. 273, Issue 33, 20744-20751, August 14, 1998

A Protein Radical and Ferryl Intermediates Are Generated by Linoleate Diol Synthase, a Ferric Hemeprotein with Dioxygenase and Hydroperoxide Isomerase Activities

Chao Su, Margareta Sahlin, and Ernst H. Oliw

From the Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden, and  Department of Molecular Biology, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden

Linoleate diol synthase (LDS) was isolated as a hemeprotein from the fungus Gaeumannomyces graminis. LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydroperoxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mainly high spin. Oxygen consumption during catalysis started after a short time lag which was reduced by 8-HPODE. Catalysis declined due to suicide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 °C showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s¹. Light absorption at 406 nm then increased at a rate of ~4 s¹, whereas the absorption at 421 nm increased after a lag time of ~5 ms at a rate of ~70 s¹. EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched after 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-HPODE 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 H synthases, ferryl intermediates and a protein radical during catalysis.

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