Characterization of heme environment and mechanism of peroxide bond cleavage in human prostacyclin synthase

Abstract

Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.

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

“…Furthermore, the residues from five substrate recognition sites in PGIS make up a highly hydrophobic environment with only two residues being hydrophilic [31]. "Truth diagram" analysis of heme EPR data indicates that both PGIS and TXAS have similar hydrophobicity in the distal heme pocket [13,14]; thus, the different volume of the heme pockets in both enzymes may be a more critical determinant for the product profile. As compared to other microsomal P450s, PGIS and TXAS take significant parts in the heterolytic cleavage, a feature of peroxidase reaction.…”

“…However, we previously reported that PGIS catalyzed 90% of homolytic and 10% of heterolytic O-O cleavage of 10-hydroperoxyoctadeca-8,12-dienoic acid [13]. In view of these facts, we expected that PGIS and TXAS would convert 15-HPETE to 15-KETE (via the homolytic cleavage and one-electron oxidation of Fe(III)) and 15-HETE (via the heterolytic cleavage and two-electron oxidation of Fe(III)) (Scheme I).…”

“…Recombinant PGIS was prepared as described previously, and its concentration was spectrophotometrically determined using ε 418 = 103 mM −1 cm −1 [13]. The TXAS expression vector was essentially the same as previously described [14] except that the amino terminal segment (MALLLAVF) was replaced by a hydrophilic segment (MAKKTSS).…”

Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change

“…1-(o-Chloro-␣,␣-diphenyl-benzyl)imidazole (clotrimazole) and 6-(1-piperidyl)-2,4-diaminopyrimidine-3-oxide (minoxidil) were from Sigma. PGH 1 and PGH 2 were synthesized as described previously (12).…”

“…In addition, the cyclization reaction is accompanied by the formation of the C-5 radical; it remains a question how this carbon radical donates an electron to Fe(IV)-porphyrin to assist the subsequent catalytic steps. Moreover, given that PGIS favors homolytic cleavage of the peroxide bond of fatty acid hydroperoxides (12,13), a reaction known to be facilitated in a nonpolar microenvironment, and that both PGH 2 and PGI 2 are hydrophobic and are labile in aqueous solution, will the structure of solvent molecules in the PGIS active site change in the presence of substrate? Finally, does the lack of direct interactions between heme propionates and protein scaffold have potential functional significance?…”