Reactions of Prostaglandin H Synthase with Monosubstituted Hydrazines and Diazenes

Abstract

The reaction of p‐chlorophenylhydrazine with prostaglandin H synthase (PGHS) Fe(III) under aerobic conditions leads to a partial destruction of the heme and to a new complex absorbing at 436 nm. This complex is also obtained by reaction of p‐chlorophenyldiazene (pClPhN = NH) with PGHS Fe(III) under anaerobic conditions and by oxidation of the PGHS Fe(II)(pClPhN = NH) diazene complex by Fe(CN)6K3. The similarity between those reactions and those of arylhydrazines and aryldiazenes with other hemoproteins such as cytochrome P450 and hemoglobin and myoglobin, as well as the similarities between the spectroscopic and chemical properties of this complex and those of the σ‐aryl complexes of other hemoproteins such as hemoglobin and myoglobin, strongly suggested a PGHS Fe(III)‐pClPh structure for this complex. It was completely established after the extraction of its heme, by butan‐2‐one at 0°C under neutral or acidic conditions, which led to the σ‐aryl PGHS‐Fe(III)‐pClPh complex and to N‐phenylprotoporphyrin IX, respectively. A mechanism is proposed for the formation of the PGHS Fe(III) pClPh complex; it includes the reduction of PGHS Fe(III) into PGHS Fe(II) with formation of the diazene pClPhN = NH. This diazene can bind to PGHS Fe(II) or be oxidized with formation of pClPh free radicals. These radicals can react with PGHS Fe(II) to form the PGHS Fe(III)‐pClPh complex or with the protein, or may initiate free radical oxidations which could lead to destruction of the heme or of the protein. Other alkylhydrazines or arylhydrazines also react with PGHS Fe(III) under aerobic conditions with the formation of PGHS Fe(III)‐R or aryl (Ar) complexes and heme destruction. Alkylhydrazines such as methylhydrazine, which lead to very reactive alkyl radicals, lead to very low amounts of PGHS Fe(III)‐R complex and high amounts of heme destruction, whereas arylhydrazines bearing electron‐withdrawing substituents such as 3,4‐dichlorophenylhydrazine, which lead to stabilized aryl radicals, lead to a high amounts of PGHS Fe(III)‐Ar complex and low amounts of heme destruction. Some information concerning the structure of the active site of PGHS has been drawn from those studies as follows: (a) the formation of σ‐aryl PGHS Fe(III)‐Ar and PGHS Fe(II)(ArN = NH) complexes bearing bulky ligands on the iron is in agreement with a large hydrophobic active site with an easily accessible iron atom and, (b) a comparison of the UV‐visible characteristics of PGHS Fe(III)‐R or Ar complexes with those of other hemoprotein‐Fe(III)‐R or hemoprotein‐Fe(III)‐Ar complexes is in agreement with a histidine as the axial proximal ligand of the iron of PGHS. Finally, the formation of iron(III)‐R or iron (III)‐Ar complexes and the free radical oxidative degradation of the heme could both explain why monosubstituted hydrazines are powerful inhibitors of PGHS and very bad reducing cosubstrates of the peroxidase function of PGHS.