Dissection of Porphyrin-Induced Conformational Dynamics in the Heme Biosynthesis Enzyme Ferrochelatase
Dissection of Porphyrin-Induced Conformational Dynamics in the Heme Biosynthesis Enzyme Ferrochelatase
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DOI：
10.1021/bi300704c
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发表时间：
2012-09-11
期刊：
影响因子：
2.9
通讯作者：
Busenlehner, Laura S.
中科院分区：
文献类型：
--
作者：
Asuru, Awuri P.;An, Mier;Busenlehner, Laura S.
人铁螯合酶（EC 4.99.1.1）催化亚铁插入原卟啉IX作为血红素生物合成的最后一步，鉴于血红素在细胞内的巨大功能，这是大多数生物体必不可少的过程。即使有多种铁螯合酶结构可用，铁插入卟啉的确切机制仍然是一个有争议的问题。然而，很明显，构象动力学对于卟啉底物结合、铁的初始螯合、铁插入大环和原血红素IX的释放是重要的。在这项工作中，我们利用底物介卟啉（MPIX）和主酰胺氢/氘交换质谱（HDX-MS）表征了与卟啉结合相关的铁螯合酶的构象和动态变化。一般来说，与卟啉直接或间接的相互作用使活性位点周围的区域变得更加有序。我们的研究结果表明，活性位点口的下唇被预先组织为有效的卟啉结合，骨干动力学几乎没有变化。上唇区域在HDX行为上有最显著的变化，因为它关闭了活性部位。这种运动将溶剂从卟啉口袋中排除，但导致溶剂在其他区域的访问增加。观察到一条通往活性位点的水通道，这可能是难以捉摸的铁通道，最终通过卟啉的M76/R164/Y165侧插入。这些结果通过包含动态信息提供了铁螯合酶机制的严格观点，揭示了功能研究的新结构区域，并为潜在的铁通道到活性位点提供了新的见解。
Human ferrochelatase (EC 4.99.1.1) catalyzes the insertion ferrous iron into protoporphyrin IX as the last step in heme biosynthesis, an essential process to most organisms given the vast intracellular functions of heme. Even with multiple ferrochelatase structures available, the exact mechanism for iron insertion into porphyrin is still a matter for debate. It is clear, however, that conformational dynamics are important for porphyrin substrate binding, initial chelation of iron, insertion of iron into the macrocycle, and release of protoheme IX. In this work we characterize conformational and dynamic changes in ferrochelatase associated with porphyrin binding using the substrate mesoporphyrin (MPIX) and backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS). In general, regions surrounding the active site become more ordered from direct or indirect interactions with the porphyrin. Our results indicate that the lower lip of the active site mouth is preorganized for efficient porphyrin binding, with little changes in backbone dynamics. The upper lip region has the most significant change in HDX behavior as it closes the active site. This movement excludes solvent from the porphyrin pocket, but leads to increased solvent access in other areas. A water lined path to the active site was observed, which may be the elusive iron channel with final insertion via the M76/R164/Y165 side of the porphyrin. These results provide a rigorous view of the ferrochelatase mechanism through the inclusion of dynamic information, reveal new structural areas for functional investigation, and offer new insight into a potential iron channel to the active site.