A protein radical and ferryl intermediates are generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and hydroperoxide isomerase activities.
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 degreesC showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s-1. Light absorption at 406 nm then increased at a rate of approximately 4 s-1, whereas the absorption at 421 nm increased after a lag time of approximately 5 ms at a rate of approximately 70 s-1. 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.
Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden.
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D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005290:Ferric Compounds / Q000737:chemistry; D005609:Free Radicals; D005979:Glutathione Peroxidase / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism
D005290:Ferric Compounds; D005609:Free Radicals; D006420:Hemeproteins; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D010105:Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase