Department of Chemistry, 1102 Natural Science II, University of California, Irvine, California 92697, United States.
Department of Chemistry, University of Basel, P.O. Box 3350, Mattenstrasse 24a, BPR 1096, CH-4002 Basel, Switzerland.
Inorg Chem. 2020 May 4;59(9):6000-6009. doi: 10.1021/acs.inorgchem.9b03791. Epub 2020 Apr 20.
An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the SE mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic Fe complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from SE. Two aqua ligands complete the primary coordination sphere of the Fe center with both involved in hydrogen bond networks within Sav. The corresponding Fe protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The Fe protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
一类重要的非血红素双加氧酶含有一个保守的 Fe 结合位点，由 2-组氨酸-1-羧酸盐面三角组成。结构生物学的结果表明，在静止状态下，这些蛋白质具有六配位，水分子占据剩余的三个配位位。我们利用生物素-链霉亲和素（Sav）技术设计了新的人工 Fe 蛋白（ArMs），这些蛋白具有这些非血红素双加氧酶活性位点中发现的许多相同的结构特征。分离到一种含有 SE 突变的 Sav 变体，该突变在适当的位置安装了一个羧酸盐侧链，以与限制在 Sav 内的合成 Fe 配合物结合。使用 X 射线衍射（XRD）方法进行的结构研究揭示了一个面三角结合位点，由生物素化配体的两个 N 供体和 SE 的单齿配位的羧酸盐组成。两个水分子完成了 Fe 中心的初级配位球，两者都参与 Sav 内的氢键网络。还制备并结构表征了相应的 Fe 蛋白，以显示具有两个外源乙酰丙酮配体的六配位络合物。通过取代一个乙酰丙酮配体，Fe 蛋白进一步被证明可以结合外源叠氮配体。溶液中 ArMs 的光谱研究支持 XRD 发现的结果。
