EPR study of ferric native prostaglandin H synthase and its ferrous NO derivative.
Purified prostaglandin H synthase (EC 1.14.99.1) apoprotein, a polypeptide of 72 kDA, was titrated with hemin and EPR spectra of high-spin ferric heme were observed at liquid-helium temperature. With up to one hemin per polypeptide, a signal at g = 6.6 and 5.4, rhombicity 7.5%, evolved owing to specifically bound, catalytic active heme. At higher heme/polypeptide ratios signals at g = 6.3 and 5.9 were observed which were assigned to non-specific heme with no catalytic function. In microsomes from ram seminal vesicles the native enzyme showed the signal at g = 6.7 and 5.2 which could not be increased by the addition of hemin. Cyanide, an inhibitor of the enzyme, reacted at lower concentrations with the specific heme abolishing its signal at g = 6.6 and 5.4. Higher concentrations of cyanide were needed for the disappearance of the signal of non-specific heme. The reduced enzyme reacted with NO and formed two types of NO complexes. A transient complex, with a rhombic signal at gx = 2.07, gz = 2.01 and gy = 1.97, was assigned to a six-coordinate complex. The final, stable complex showed an axial signal at g = 2.12 and g = 2.001 and was assigned to a five-coordinate complex, where the protein ligand was no longer bound to the heme iron. Neither type of signal showed a hyperfine splitting from nitrogen of histidine indicating the absence of a histidine-iron bond in the enzyme. From these results and the similarity of the EPR signal at g = 6.6 and 5.4 to the signal of native catalase (EC 1.11.1.6) we speculated that tyrosinate might be the endogenous ligand of the heme in prostaglandin H synthase.
D000818:Animals; D002384:Catalysis; D003486:Cyanides; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000032:analysis; D005296:Ferrous Compounds / Q000032:analysis; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000032:analysis*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009569:Nitric Oxide; D010084:Oxidation-Reduction; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep
D003486:Cyanides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D009569:Nitric Oxide; D006418:Heme; D006427:Hemin; D011451:Prostaglandin-Endoperoxide Synthases