INFRARED SPECTROSCOPIC STUDIES OF CARBONYL HORSERADISH PEROXIDASES
INFRARED SPECTROSCOPIC STUDIES OF CARBONYL HORSERADISH PEROXIDASES
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DOI：
10.1021/bi00655a031
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
PAUL, KG
中科院分区：
文献类型：
--
作者：
BARLOW, CH;OHLSSON, PI;PAUL, KG
辣根过氧化物酶同工酶 A2 和 C 的红外差异光谱（FeIICO 与 FeIII）在 2000-1800 cm-1 范围内记录。在碱性条件下，pH 9，两种同工酶都显示出 2 个 CO 伸缩带，A2 在 1938 和 1925 cm-1，C 在 1933 和 1929 cm-1。随着 pH 值降低，每种同工酶的低频带强度降低，同时出现 A2 和 C 的 1906 和 1905 cm-1 谱带，强度增加分别是同工酶。这些变化符合辣根过氧化物酶 A2 同工酶的 pK 值 6.7 和 C 同工酶的 pK 值 8.8。通过观察 FeIII/FeII 系统的氧化还原电位 Em7 与细胞色素 a3、Hb、肌红蛋白和细胞色素 P-450cam 与底物的 IR CO 拉伸频率 .nu.CO 之间存在线性关系，简化了对 IR 结果的解释。改变这些血红素蛋白中 .nu.CO 的主要力量显然是血红素 Fe 处电子密度的变化，而不是与 CO 配体的直接蛋白质相互作用。 1930-cm-1 区域的辣根过氧化物酶 IR 谱带与这种关系密切相关。 1905-cm-1带与线性关系的较大偏差及其对氢离子浓度的依赖性与具有单个CO结合位点的辣根过氧化物酶一致，该结合位点可以保持2种几何形状，其中一种包含能够与羰基氧形成H键的氨基酸部分。
IR difference spectra, FeIICO vs. FeIII, of horseradish peroxidase isoenzymes A2 and C were recorded from 2000-1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibited 2 CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH was lowered, the low-frequency band for each isoenzyme decreased in intensity with a concommitant appearance and increase in intensity of a band of 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conformed to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the IR results was simplified by the observation that a linear relationship existed between the redox potential, Em7, for the FeIII/FeII system vs. the IR CO stretching frequency, .nu.CO, for cytochrome a3, Hb, myoglobin and cytochrome P-450cam with substrate. The primary force altering .nu.CO in these heme proteins is apparently a variation in electron density at the heme Fe and not direct protein interactions with the CO ligand. The horseradish peroxidase IR bands in the 1930-cm-1 region correlated well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration were consistent with horseradish peroxidase having a single CO binding site which can hold in 2 geometries, one of which contained an amino acid moiety capable of forming a H bond to the carbonyl oxygen.