Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines.

Various N-alkylhydroxylamines such as N-hydroxyamphetamine react with prostaglandin synthase (PGHS) from sheep seminal vesicles, with the formation of new complexes characterized by a Soret peak around 421 nm. These complexes are very stable toward O2 or dithionite but are destroyed upon oxidation by Fe(CN)6K3 with regeneration of starting PGHS-FeIII. Their spectral characteristics, chemical properties, and routes of formation (either by direct oxidation of RNHOH or by in situ reduction of RNO2 in the presence of dithionite) are very similar to those previously reported for nitrosoalkane complexes of hemoglobin-, myoglobin-, and cytochrome P-450-FeII. Their FeII-N(O)R structure was completely confirmed in the case of N-hydroxyamphetamine, both by extraction of the heme complex by butanone and by identification to authentic protoporphyrin IX-FeII-N(O)-amphetamine, and by insertion of this authentic complex into apoPGHS. Phenylhydroxylamine also reacts with PGHS-FeIII to give a PGHS-FeII-N(O)Ph complex which is not stable in the presence of dithionite because of its weaker PGHS-FeII-N(O)R bond when compared to PGHS-FeII-nitrosoalkane complexes. The ability of various N-alkylhydroxylamines to form PGHS-FeII-N(O)R complexes greatly depends upon their hydrophobicity. Actually, CH3NHOH and C2H5NHOH are totally inactive whereas about 10 molar excess of N-hydroxyamphetamine and C6H5NHOH already lead to 50% complex formation. This is in favor of an hydrophobic environment of the heme in PGHS. Finally, PGHS engaged in such FeII-nitrosoalkane complexes completely loses its dioxygenase activity, suggesting that N-substituted hydroxylamines or compounds that can be metabolized in vivo to give such hydroxylamines could act as strong PGHS inhibitors.