The mechanisms of lipoxygenase inhibition by iron chelators have been investigated in human neutrophils and in isolated soybean lipoxygenase. Their Fe(III)-containing active sites have been targeted by synthesizing novel bidentate chelators from the hydroxypyridinone family sufficiently small to gain access through the hydrophobic channels of lipoxygenase. In stimulated human neutrophils, release of [3H]arachidonate-labeled eicosanoids is dependent on the lipid solubility of hydroxypyridinones, but larger hexadentate chelators have no effect on this or on total cellular leukotriene B4 production. Lipophilic hydroxypyridinones inhibit 5-lipoxygenase at equivalent concentrations to the established inhibitor, piriprost, and show additional but minor anti-phospholipase A2 activity. Soybean 15-lipoxygenase inhibition is also dependent on the lipid solubility and coordination structure of chelators. Inhibition is associated with the formation of chelate-iron complexes, which are removed by dialysis without restoration of enzyme activity. Only after adding back iron is activity restored. Electron paramagnetic resonance studies show the removal of the iron center signal (g = 6) is concomitant with formation of Fe(III)-chelator complexes, identical in spectral shape and g value to 3:1 hydroxypyridinone Fe(III) complexes. Removal of iron is not the only mechanism by which hydroxypyridinones can inhibit lipoxygenase in intact cells, however, as a lipophilic non-iron-binding hydroxypyridinone, which shows no inhibition of the soybean lipoxygenase activity, partially inhibits 5-lipoxygenase in intact neutrophils without inhibiting neutrophil phospholipase A2.

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G 2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG 2 to PGH 2 . The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr 385 to a tyrosyl radical within the cyclooxygenase site. His 386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr 385 , which protrudes into the cyclooxygenase site, and His 388 , the proximal ligand of the peroxidase heme. When His 386 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- and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.

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