Study of the coordination chemistry of prostaglandin G/H synthase by resonance Raman spectroscopy

Resonance Raman spectra of prostaglandin G/H synthase (PGHS) in its ferric and ferrous states have been obtained by Soret excitation. In native PGHS, which contained only 0.25 heme/monomeric apoprotein, the ferric heme was in a high-spin hexacoordinated state. The presence of a vibration at 489 cm(-1) that was responsive to (H2O)-O-16-->(H2O)-O-18 replacement was taken as evidence for the presence of a H-bonded H2O molecule as the sixth ligand of the Fe. A study by CD and resonance Raman spectroscopy, of heme incorporation into the apoprotein showed that, for heme/protein ratios lower than 0.5, the heme was in the same ferric high-spin hexacoordinated state as in the native enzyme. For heme/protein ratios higher than 0.5, the concomitant formation of two minor species was observed: a low-spin hexacoordinated species which could be due to the axial coordination of a distal histidine to the Fe trans to its proximal histidine ligand; and a high-spin pentacoordinated species that corresponded to non-specific binding of the heme to the apoprotein. In the reduced state, the heme of PGHS contained a high-spin pentacoordinated Fe(II) with a histidine as the proximal ligand. However, this species shifted spontaneously towards a low-spin hexacoordinated Fe(II) species in which the iron was probably coordinated by a distal histidine as the sixth axial ligand. The PGHS Fe(II) CO derivative displayed an Fe-CO stretching mode at 529 cm(-1), which is in the range observed for peroxidases. Such a high frequency could be due to H-bonding between the oxygen atom of the CO ligand and the distal histidine, His207. Since this histidine plays an important role, by coordination of Fe(II) or Fe(III) of PGHS and stabilization of the ligands of the Fe, H2O or CO by H-bonding, it is suggested that this histidine could also play a key role in the cleavage of the O-O bond of peroxides by peroxidases.