Gaspard S, Chottard G, Mahy J P, Mansuy D
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université Paris V, France.
Eur J Biochem. 1996 Jun 1;238(2):529-37. doi: 10.1111/j.1432-1033.1996.0529z.x.
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 289 cm-1 that was responsive to H(2)16O -> H(2)18O 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.
通过索雷特激发获得了处于三价铁和二价铁状态的前列腺素G/H合酶（PGHS）的共振拉曼光谱。在仅含有0.25个血红素/单体脱辅基蛋白的天然PGHS中，三价铁血红素处于高自旋六配位状态。289 cm-1处存在对H(2)16O -> H(2)18O置换有响应的振动，这被视为存在一个以氢键结合的水分子作为铁的第六个配体的证据。一项通过圆二色光谱和共振拉曼光谱对血红素掺入脱辅基蛋白的研究表明，对于血红素/蛋白质比率低于0.5的情况，血红素与天然酶中的三价铁高自旋六配位状态相同。对于血红素/蛋白质比率高于0.5的情况，观察到同时形成了两种次要物种：一种低自旋六配位物种，这可能是由于一个远端组氨酸与铁的轴向配位，该铁与其近端组氨酸配体呈反式；以及一种高自旋五配位物种，它对应于血红素与脱辅基蛋白的非特异性结合。在还原状态下，PGHS的血红素含有一个高自旋五配位的Fe(II)，其近端配体为组氨酸。然而，该物种会自发地转变为低自旋六配位的Fe(II)物种，其中铁可能由一个远端组氨酸作为第六个轴向配体配位。PGHS Fe(II).CO衍生物在529 cm-1处显示出Fe-CO伸缩模式，这在过氧化物酶观察到的范围内。如此高的频率可能是由于CO配体的氧原子与远端组氨酸His207之间的氢键作用。由于该组氨酸通过PGHS的Fe(II)或Fe(III)的配位以及通过氢键对Fe的配体H2O或CO的稳定作用发挥重要作用，因此有人提出该组氨酸在过氧化物酶催化过氧化物的O - O键断裂中也可能起关键作用。
