Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase -Formation of an N-hydroxylamine

Synthesis of prostaglandin H-2 by prostaglandin H synthase (PHS) results in a two electron oxidation of the enzyme. An active reduced enzyme is regene rated by reducing cofactors, which become oxidized. This report examines th e mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conv ersion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphe nyl, a new metabolite was observed when 1 mM ascorbic acid was present. Sim ilar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 w as used as the substrate. This metabolite co-eluted with synthetic N'-hydro xy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'H A represented as much as 10% of the total radioactivity recovered by high p ressure liquid chromatography. When N'HA was substituted for ABZ, PHS metab olized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrat ed that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase su ggests the lack of involvement of one-electron transfer reactions and sugge sts that hydroxyl radicals and superoxide are not sources of oxygen or oxid ants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [O-18]H2O2 was used as the substrate, O-18 enrichment was obs erved for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichmen t was observed for one atom of O-18, and a 17 +/- 7% enrichment was observe d for two O-18 atoms. The rapid exchange of O-18-N'HA with water was sugges ted to explain the lack of enrichment of N'HA and the low enrichment of two O-18 atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a pero xygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.