Heme coordination of prostaglandin H synthase.

A.L Tsai,R.J Kulmacz,J.S Wang,Y Wang,H.E Van Wart,G Palmer

doi:10.1016/s0021-9258(18)52911-2

A.L Tsai, R.J Kulmacz + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)52911-2

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Abstract

The heme coordination of ovine prostaglandin H synthase (PGHS) has been characterized by EPR, magnetic circular dichroism, resonance Raman, and optical spectroscopies. The EPR spectrum of ferric PGHS is consistent with an equilibrium mixture of high-spin and low-spin heme species. Both species disappear on reaction of the synthase with hydroperoxides. The high-spin to low-spin interconversion is temperature- and concentration-dependent. Correlation between the axial and rhombic ligand fields of the low-spin heme species suggests that it has bishistidine axial ligation. Magnetic circular dichroism spectra of PGHS also show a temperature-dependent spin transition. Resonance Raman spectra indicate that the enzyme exists as a mixture of six-coordinate low-spin and six-coordinate high-spin ferric heme species. No Raman bands attributable to five-coordinate high-spin heme species are detectable. The magnetic circular dichroism spectra of the fluoride, azide, cyanide, and imidazole derivatives of PGHS resemble those of the corresponding metmyoglobin derivatives and are very different from those of the catalase derivatives. EPR spectra of the imidazole derivative of these three proteins provide additional evidence that the heme coordination structure of PGHS is similar to that of metmyoglobin rather than that of catalase. The midpoint potential of the PGHS Fe(III)/Fe(II) pair is in the range observed for hemeproteins with mono- or bishistidine coordination. These data provide a convincing case that the axial heme ligands of PGHS-1 are a pair of histidine residues, with the distal histidine weakly associated and possibly exchangeable with a weak-field ligand.

Highlights

From the $Division of Hematology, Department of Internal Medicine, University of Teras Health Science Center, Houston, Texas 77030, the lllmtitute of Molecular Biophysics, Florida State Uniuersity, Tallahsee, Florida 32306, and the
Correlation between the of the profound influence of the axial ligands on the catalytic axial and rhombic ligand fields of the low-spin heme actions of heme in hemeproteins, a molecular interpretation species suggests that it has bishistidine axialligation. of the reaction mechanisms of Prostaglandin H synthase (PGHS) requires information
Attributable to five-coordinatehigh-spin heme species proposed that tyrosine is the proximal heme ligand, based on are detectable

Summary

Heme Coordination of Prostaglandin H Synthase*

EPR spectra of the imidazole derivative of these three proteins provide additional ing histidine as proximal heme ligand, e.g. cytochrome-c perevidence that the heme coordination structure of PGHS oxidase and myeloperoxidase, are known to have a rhombicity is similar to thatof metmyoglobin rather than thatof similar to thatof the high-spin heme in PGHS [6, 7], and so catalase. A comparison of the electronic spectra of metmyoglobin removed during the titration and diluted 1:lOOO to measure (with a histidine proximal ligand), catalase I. All three hemeproteins exhibit a ligand-to-iron or porphy- (Fig. 3), indicating that imidazole binding was reversible and rin-to-iron charge-transfer band at 600-650 nm that is char- that the prosthetic group had not been displaced from the acteristic of high-spin heme species.in terms of protein by the ligand. Eachsample was component with reference to a bisimidazole myoglobin standis present half of the heme in resting PGHS prepared in 50 mM potassium phosphate (pH 7.2) containing 0.02% ard indicates that octyl @-D-glucoside

Native or aquo

PGH Synthase d

PCH Synthase z

This study

The resonance Raman spectra of resting PGHS indicate a