Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species.
Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species.
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DOI：
10.1021/bi051902l
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发表时间：
2006-01
期刊：
影响因子：
2.9
通讯作者：
Benoit J. Smagghe;G. Sarath;E. Ross;J. Hilbert;M. Hargrove
中科院分区：
文献类型：
--
作者：
Benoit J. Smagghe;G. Sarath;E. Ross;J. Hilbert;M. Hargrove
从原核生物到植物和动物，在许多生物中都发现了六配位血红蛋白。由于位于血红素口袋中的组氨酸侧链对血红素铁的可逆配位作用，它们被命名为“六坐标”。这种内源性配位与外源性配体结合竞争，并在快速混合或闪光光解实验后引起多相驰豫时间过程。以前的快速混合研究假设了六配位和外源配体结合之间的稳态关系，而不是与观察到的结合时间进程相关。在这里，我们证明了这一假设对某些六配位血红蛋白是不成立的，并且多相时间过程是由于相当一部分五坐标血红素是由相对较小的六配位平衡常数(K(H))引起的。测定了四种植物六配位血红蛋白、人脑红蛋白和细胞球蛋白以及聚球藻血红蛋白的快速混合引发的CO结合反应。植物蛋白质虽然表现出惊人的变异性，但与其他蛋白质的不同之处在于K(H)值要低得多。脑红蛋白和细胞红蛋白显示了CO结合的戏剧性的双相时间过程，这是使用其他技术所没有观察到的。最后，提出了一种独立的K(H)光谱定量方法，以补充用于六配位研究的快速混合。这些结果表明，与植物六配位血红蛋白相比，六配位配位在调节人脑红蛋白和细胞球蛋白与配体结合的亲和常数方面可能起到更大的作用。
Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.