Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

中文翻译：

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洞察活性位点残基在人亚铁螯合酶催化机制中的功能

铁螯合酶催化亚铁插入卟啉大环以产生必需的辅因子血红素。在人类中，这种酶不仅催化末端步骤，而且在血红素合成途径中起到调节步骤的作用。人类亚铁螯合酶的十多种晶体结构已被解析，许多变体已被动力学表征。此外，氢氘交换、共振拉曼、分子动力学和高水平量子力学研究增加了我们对酶催化循环的理解。然而，了解金属离子如何传递以及活性位点残基在催化中的具体作用仍然是悬而未决的问题。数据与发生在吡咯质子提取相反一侧的金属结合和插入一致。为了更好地了解铁的传递和结合以及活性位点中保守残基的作用，我们构建并表征了一系列酶变体。进行了晶体学研究以及变体的拯救和动力学分析。这些研究的数据与 M76 残基在活性位点金属结合和形成产品释放所必需的弱铁蛋白配体中发挥作用一致。此外，结构数据支持 E343 在质子提取和产品释放中与由 Q302、S303 和 K304 组成的肽环协调作用，这些肽环充当金属传感器。进行了晶体学研究以及变体的拯救和动力学分析。这些研究的数据与 M76 残基在活性位点金属结合和形成产品释放所必需的弱铁蛋白配体中发挥作用一致。此外，结构数据支持 E343 在质子提取和产品释放中与由 Q302、S303 和 K304 组成的肽环协调作用，这些肽环充当金属传感器。进行了晶体学研究以及变体的拯救和动力学分析。这些研究的数据与 M76 残基在活性位点金属结合和形成产品释放所必需的弱铁蛋白配体中发挥作用一致。此外，结构数据支持 E343 在质子提取和产品释放中与由 Q302、S303 和 K304 组成的肽环协调作用，这些肽环充当金属传感器。