Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily.
Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily.
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SznF（血红素加氧酶样二铁氧化酶/加氧酶超家族成员）的生物物理表征方法。
DOI：
10.1007/978-1-0716-3080-8_9
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Boal,AmieK
中科院分区：
文献类型：
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作者：
McBride,MollyJ;Pope,SarahR;Nair,MrutyunjayA;Sil,Debangsu;Salas-Solá,XavierE;Krebs,Carsten;MartinBollingerJr,J;Boal,AmieK
非血红素二铁酶利用氧的化学势来催化生物学中具有挑战性的反应。在其静止状态下，这些酶具有由组氨酸和羧酸配体配位的二铁辅因子。在暴露于氧气时，辅因子氧化至其二铁态，形成过氧加合物，能够催化宽范围的氧化化学反应，例如去饱和和杂原子氧化。尽管它们的多功能性和威力，一个新兴的子集的非血红素二铁酶具有固有的辅因子不稳定性，使它们耐结构表征。这一特征在血红素加氧酶样二铁氧化酶/加氧酶（HDO）超家族的成员中广泛存在。HDO具有在金属结合时重塑的柔性芯结构。虽然已经出土了约9600个HDO，但迄今为止很少有功能性特征。在本章中，我们描述了已被用来表征HDON加氧酶，SznF的方法。我们展示了apo-SznF的过表达和纯化以及专门设计用于帮助获得全息-SznF的X射线结构的方法。我们还描述了表征的瞬态SznF-peroxo-Fe（III）2复杂的停流吸收和穆斯堡尔谱。这些研究为HDO超家族新成员的表征提供了框架。
Nonheme diiron enzymes harness the chemical potential of oxygen to catalyze challenging reactions in biology. In their resting state, these enzymes have a diferrous cofactor that is coordinated by histidine and carboxylate ligands. Upon exposure to oxygen, the cofactor oxidizes to its diferric state forming a peroxo- adduct, capable of catalyzing a wide range of oxidative chemistries such as desaturation and heteroatom oxidation. Despite their versatility and prowess, an emerging subset of nonheme diiron enzymes has inherent cofactor instability making them resistant to structural characterization. This feature is widespread among members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily. HDOs have a flexible core structure that remodels upon metal binding. Although ~9600 HDOs have been unearthed, few have undergone functional characterization to date. In this chapter, we describe the methods that have been used to characterize the HDON-oxygenase, SznF. We demonstrate the overexpression and purification of apo-SznF and methodology specifically designed to aid in obtaining an X-ray structure of holo-SznF. We also describe the characterization of the transient SznF-peroxo-Fe(III)2complex by stopped-flow absorption and Mössbauer spectroscopies. These studies provide the framework for the characterization of new members of the HDO superfamily.