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

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 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-δ of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-δ 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.