Distal Regulation of Heme Binding of Heme Oxygenase-1 Mediated by Conformational Fluctuations
Distal Regulation of Heme Binding of Heme Oxygenase-1 Mediated by Conformational Fluctuations
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DOI：
10.1021/bi5009694
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发表时间：
2015-01-20
期刊：
影响因子：
2.9
通讯作者：
Sugase, Kenji
中科院分区：
文献类型：
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作者：
Harada, Erisa;Sugishima, Masakazu;Sugase, Kenji
血红素加氧酶-1（HO-1）是一种催化血红素氧化降解的酶。由于游离血红素对细胞有毒，血红素的快速降解对于维持细胞健康是重要的。已经有基于晶体结构的HO反应的有用的机理研究;然而，HO-1如何识别血红素还没有完全理解，因为无血红素的大鼠HO-1的晶体结构缺乏连接血红素的A-螺旋的电子密度。在这项研究中，我们的特点是构象动力学的HO-1使用NMR阐明的机制，HO-1识别血红素。核磁共振弛豫实验表明，血红素结合位点在血红素自由HO-1波动与表面暴露的环，并短暂地形成一个部分展开的结构。由于波动环位于血红素结合位点的17埃远侧，并且其构象在不同的晶体结构（包括催化中间态）中几乎相同，因此环的功能尚未被研究。在阐明其功能的过程中，我们发现了这个环中有趣的突变，这些突变改变了活性，但对构象几乎没有影响。环中的Phe 79 Ala突变改变了血红素结合位点的构象动力学。此外，突变体的血红素结合动力学比野生型慢。因此，我们的结论是远端环参与的血红素结合通过构象波动的构象变化的调节。与其他酶类似，HO-1利用所鉴定的远端位点有效地促进其功能，这可能是蛋白质工程的潜在靶点。
Heme oxygenase-1 (HO-1) is an enzyme that catalyzes the oxidative degradation of heme. Since free heme is toxic to cells, rapid degradation of heme is important for maintaining cellular health. There have been useful mechanistic studies of the HO reaction based on crystal structures; however, how HO-1 recognizes heme is not completely understood because the crystal structure of heme-free rat HO-1 lacks electron densities for A-helix that ligates heme. In this study, we characterized conformational dynamics of HO-1 using NMR to elucidate the mechanism by which HO-1 recognizes heme. NMR relaxation experiments showed that the heme-binding site in heme-free HO-1 fluctuates in concert with a surface-exposed loop and transiently forms a partially unfolded structure. Because the fluctuating loop is located over 17 angstrom distal from the heme-binding site and its conformation is nearly identical among different crystal structures including catalytic intermediate states, the function of the loop has been unexamined. In the course of elucidating its function, we found interesting mutations in this loop that altered activity but caused little change to the conformation. The Phe79Ala mutation in the loop changed the conformational dynamics of the heme-binding site. Furthermore, the heme binding kinetics of the mutant was slower than that of the wild type. Hence, we concluded that the distal loop is involved in the regulation of the conformational change for heme binding through the conformational fluctuations. Similar to other enzymes, HO-1 effectively promotes its function using the identified distal sites, which might be potential targets for protein engineering.