Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases.

Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are members of the cytochrome P450 superfamily, which uses heme as the prosthetic group and has a cysteine thiolate proximal ligand [1, 2]. PGIS and TXAS convert prostaglandin H2 (PGH2) to prostacyclin (PGI2) and thromboxane A2 (TXA2), respectively. PGI2 inhibits platelet activation and aggregation and induces vasodilation, whereas TXA2 stimulates platelet secretion, aggregation and vasoconstriction [3]. Both PGI2 and TXA2 are rapidly hydrolyzed to biologically inactive forms, thus making PGI2 and TXA2 autocrine or paracrine mediators. PGIS and TXAS are localized to the endoplasmic reticulum membrane. Unlike other microsomal P450s, they catalyze isomerization rather than mono-oxygenation reactions. In addition, they undergo suicide inactivation during catalysis. Although PGIS shares many features with TXAS, they only have 16% sequence identity, suggesting a potential difference in reaction mechanisms. Hecker and Ullrich proposed a caged radical mechanism for TXAS and PGIS catalysis [4]. In this mechanism, the initial step of substrate binding and interaction of one of the endoperoxide oxygens of PGH2 with the ferric heme iron is followed by homolytic scission of the peroxide bond, leading to formation of alkoxy radical and [S−⋯Fe(IV)⋯O–R] intermediates. With TXAS, the alkoxy radical undergoes a β-scission and ionic rearrangement to form TXA2. With PGIS, the alkoxy radical undergoes cyclization, giving rise to a carbon-centered radical. Subsequent oxidation of the carbon-centered radical and formation of an alkene generate PGI2. Both TXAS and PGIS utilize an endoperoxide (PGH2) as their substrate, and some aspects of peroxidase-based mechanisms may be relevant. Most peroxidases have heme Fe(III) as the prosthetic group and catalyze a two-electron oxidation to produce an intermediate called Compound I (Cpd I) [5]. Subsequent one-electron reduction of Cpd I generates Compound II (Cpd II), which contains an Fe(IV)-oxo complex. A further one-electron reduction regenerates the resting peroxidase. Hydroperoxides have been frequently used in studying P450 reaction mechanisms because they provide an effective alternative for the separate addition of molecular oxygen and reducing equivalents, the so-called “peroxide shunt”. Numerous reports indicated that P450s catalyze both homolytic and heterolytic cleavages of hydroperoxides and peroxyacids [6-8]. Homolytic O–O scission produces Cpd II and an alkoxyl radical (RO·) (eq. [1]); the alkoxyl radical is subsequently oxidized to a carbonyl product or subjected to β-scission. Heterolytic O–O scission yields an alcohol (ROH) and Cpd I, which is a complex of Fe(IV)=O with either a porphyrin radical or an amino acid radical, [X]·+ (eq. [2]). ROOH+Fe(III)→RO⋅+Fe(IV)=O [1] ROOH+Fe(III)→ROH+[Por]⋅+⋯Fe(IV)=Oor[X]⋅+⋯Fe(IV)=O [2] 15-HPETE is a 20-carbon fatty acid hydroperoxide derived from arachidonic acid and is a potential substrate for both PGIS and TXAS. In mammals, 15-HPETE can be synthesized by 15-lipoxygenase present in various tissues and cells [9]. Weiss et al first reported product analysis of 15-HPETE reacted with P450-enriched liver microsomes isolated from phenobarbital-treated rats [10]; the main products were 13-OH-14,15-EET (an epoxyalcohol) and 11,14,15-trihydroxy-5,8,12-trienoic acid. Later, purified P450 2B1 was reported to convert 15-HPETE to11-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid and, to a lesser extent, 13-OH-14,15-EET [11]. It was postulated that 15-HPETE was first homolytically cleaved by heme iron(III) to generate a ferryl-hydroxo complex and an epoxy allylic radical. The enzyme then returns to the ferric state by donating the hydroxyl radical to the allylic radical, producing an epoxyalcohol. Interestingly, 15-KETE and 15-HETE were not detected as products from these typical microsomal P450s. To understand how the reactivities and mechanisms of PGIS and TXAS differ from other P450s, we analyzed the products of PGIS and TXAS reactions with 15-HPETE. The results indicate that PGIS and TXAS convert 15-HPETE to a mixture of products comprised of 15-HETE, 15-KETE, and an epoxyalcohol.