Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization.
Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization.
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DOI：
10.1016/s0021-9258(18)67355-7
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发表时间：
1986-08
期刊：
影响因子：
--
通讯作者：
S. Hashimoto;J. Teraoka;T. Inubushi;T. Yonetani;T. Kitagawa
中科院分区：
文献类型：
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作者：
S. Hashimoto;J. Teraoka;T. Inubushi;T. Yonetani;T. Kitagawa
用Soret谱带研究了铁、铁细胞色素c过氧化物酶和化合物ES的共振拉曼光谱及其pH依赖性。化合物ES的Fe(IV) = O拉伸拉曼线在767 cm-1附近被分配到一个宽频带，在18O取代后转移到727 cm-1。化合物ES在H218O中有18o同位素频移，但在H216O中没有。这清楚地表明在Fe(IV) = O血红素和散装水之间发生了氧交换。与辣根过氧化物酶化合物II相比，在D2O中Fe(IV) = O拉伸拉曼带强度和频率明显高于H2O，但其频率在pH 4 ~ 11之间没有变化。Fe(II)-组氨酸拉伸拉曼线是根据54Fe同位素取代所观察到的频移来确定的。通过对该波段的强度分析，确定了铁细胞色素c过氧化物酶血红素连接电离的pKa为7.3。铁色素c过氧化物酶的拉曼光谱表明，血红素处于5位和6位高自旋结构之间的平衡状态。在中性pH下偏向于5位结构，但在pKa = 5.5过渡的酸性侧，6位血红素占主导地位。当pH值为6时，F-与血红素铁结合，而Cl-仅在酸性条件下才与血红素铁结合。当pH值为4.3时，HNO3、H2SO4、CH3COOH、HBr或HI的酸化作用导致5-和6-配位形式的种群有所不同。由此推断，在pH值为6时，被认为占据血红素铁第六个配位的水分子并没有与血红素铁配位，但pKa = 5.5残基的质子化引起了明显的结构变化，使得水分子能够与血红素铁配位。
Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.