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J. Biol. Chem., Vol. 282, Issue 25, 18233-18244, June 22, 2007

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Prostaglandin Endoperoxide H Synthases

PEROXIDASE HYDROPEROXIDE SPECIFICITY AND CYCLOOXYGENASE ACTIVATION^*

Jiayan Liu, Steve A. Seibold, Caroline J. Rieke^¶, Inseok Song^¶, Robert I. Cukier, and William L. Smith^¶1

From the Department of Biochemistry and Molecular Biology and Department of Chemistry, Michigan State University, East Lansing, Michigan 48824 and ^¶Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O₂ to prostaglandin G₂ (PGG₂). PGHS peroxidase (POX) activity reduces PGG₂ to PGH₂. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H₂O₂, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H₂O₂ or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG₂. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG₂ binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H₂O₂, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

Received for publication, February 9, 2007 , and in revised form, April 26, 2007.

^* This work was supported in part by National Institutes of Health Grant GM68848. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 5301 Medical Science Research Bldg. III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0606. Tel.: 734-647-6180; Fax: 734-764-3509; E-mail: smithww{at}umich.edu.

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