Water channel of horseradish peroxidase studied by the charge-transfer absorption band of ferric heme
Water channel of horseradish peroxidase studied by the charge-transfer absorption band of ferric heme
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DOI：
10.1021/jp037664q
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发表时间：
2004-05
影响因子：
3.3
通讯作者：
B. Zelent;A. Kaposi;Nathaniel V Nucci;K. Sharp;S. Dalosto;W. W. Wright-W.;J. Vanderkooi
中科院分区：
文献类型：
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作者：
B. Zelent;A. Kaposi;Nathaniel V Nucci;K. Sharp;S. Dalosto;W. W. Wright-W.;J. Vanderkooi
辣根过氧化物酶的血红素埋在蛋白质中，但蛋白质表面的通道将水溶液连接到血红素位点。辣根过氧化物酶铁在640 nm处有一个吸收带，这是由于卟啉环的π电子的2u HOMO和铁离子的dxy/dyz轨道之间的电荷转移（CT）跃迁。由于水通道延伸到Fe，因此CT带似乎对蛋白质的水合作用敏感。为了研究这一前提，将蛋白质掺入海藻糖/蔗糖玻璃中，并改变糖玻璃的水合作用。在10-300 K温度范围内测定了HRP在糖玻璃和甘油/水溶液中的吸收光谱。CT吸收带为电子振动精细结构。在水合糖和甘油/水中峰位置相同，但在干燥的糖玻璃中峰位置发生变化。这些数据表明，在水合但不干燥的糖玻璃中，水保留在血红素口袋中。竞争性抑制剂苯并异羟肟酸与蛋白质的结合增加了CT吸收和分辨率。使用密度泛函理论和分子力学的组合计算的苯并异羟肟酸对Fe的影响是使自旋状态3 /2相对于5 /2稳定。在低温下，对于甘油/水（在150 K下的玻璃化转变）中的蛋白质，CT带中的线的宽度比海藻糖/蔗糖（在65 °C下的玻璃化形成）中的窄。这表明CT谱带不均匀地变宽并且对溶剂敏感。CT吸收的光谱变窄发生随着温度降低在研究的温度范围内。水，如OH伸缩所示，也在这个范围内移动。研究结果进行了讨论，如何埋水和附近的收费可以调节血红素的活性。
The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640 nm that is attributed to a charge-transfer (CT) transition between the a 2u HOMO of ﷿ electrons of the porphyrin ring and the dxy/dyz orbital of the ferric ion. Because the water channel extends to the Fe, it seems likely that the CT band will be sensitive to the hydration of the protein. To study this premise, the protein was incorporated into trehalose/sucrose glasses and the hydration of the sugar glasses was varied. Absorption spectra of HRP in sugar glasses and in glycerol/water were taken in the range 10-300 K. The CT absorption band shows vibronic fine structure. The peak positions are the same in hydrated sugar and glycerol/water but the peak positions change in desiccated sugar glass. The data suggest that in hydrated, but not desiccated, sugar glass, water is retained in the heme pocket. Binding of the competitive inhibitor benzohydroxamic acid to the protein increases the CT absorption and resolution. The effect of benzohydroxamic acid on the Fe as calculated using a combination of density functional theory and molecular mechanics is to stabilize the spin state 3 /2 with respect to 5 /2. At low temperature the widths of the lines in the CT band are narrower for the protein in glycerol/water (glass transition at 150 K) than in trehalose/sucrose (glass formation at 65 °C). This indicates that the CT band is inhomogeneously broadened and sensitive to the solvent. The spectral narrowing of the CT absorption occurs as the temperature decreases over the temperature range studied. Water, as indicated by the OH stretch, also shifts in this range. The findings are discussed in terms of how buried water and nearby charges can modulate the activity of the heme.