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J. Biol. Chem., Vol. 278, Issue 46, 46163-46170, November 14, 2003

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Histidine 386 and Its Role in Cyclooxygenase and Peroxidase Catalysis by Prostaglandin-endoperoxide H Synthases^*

Steve A. Seibold, Terry Ball, Linda C. Hsi, Denise A. Mills, Rajeewa D. Abeysinghe, Renee Micielli, Caroline Jill Rieke¶, Robert I. Cukier, and William L. Smith¶||

From the ^¶Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 and the Departments of Biochemistry and Chemistry, Michigan State University, East Lansing, Michigan 48824

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G₂ from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG₂ to PGH₂. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr³⁸⁵ to a tyrosyl radical within the cyclooxygenase site. His³⁸⁶ of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr³⁸⁵, which protrudes into the cyclooxygenase site, and His³⁸⁸, the proximal ligand of the peroxidase heme. When His³⁸⁶ was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His³⁸⁶ helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N- of Asn³⁸² of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N- of His³⁸⁶ and the backbone carbonyl oxygens of Asn³⁸² and Gln³⁸³. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly⁵²⁶ and Ser⁵³⁰ and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.

Received for publication, June 16, 2003 , and in revised form, August 27, 2003.

^* This work was supported in part by Program Project Grants P01 GM57323 and R01 GM068848 from the National Institutes of Health. 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.

^|| To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 5416 Medical Science I, 1301 Catherine St., Ann Arbor, MI 48109-0606. Tel.: 734-647-6180; Fax: 734-764-3509; E-mail: smithww{at}umich.edu.

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