STRUCTURAL STUDIES OF COMPLEX IRON-SULFUR FLAVOPROTEINS

三甲胺脱氢酶(TMADH)的分子结构 对甲基营养细菌W3A1进行了X射线衍射法研究。TMADH 是一种由147,000道尔顿组成的复杂的铁-硫黄素蛋白 不同的亚基，每个约75,000道尔顿。它包含一个Fe4S4 每个分子有一个簇和一个共价结合的FMN辅酶。这种酶 催化三甲胺氧化成二甲胺和甲醛 随后将电子转移到人工受体吩嗪 甲硫酸盐。当被底物还原时，该酶表现出很强的强度 约化余因之间的自旋耦合伴随着一个明显的 构象变化。TMADH晶体为单斜晶系，空间群为P21 晶胞尺寸a=147.63A，b=71.96A，c=83.66A，Beta=97.64A。这个 晶体的衍射率达到2.0A分辨率。结构将通过以下方式进行分析 同构替换法首先在6.0A，然后在2.4A 决心。将研究6.0A地图以确定域和 酶的亚基结构。本机异常散射数据将是 分析确定了铁硫团的位置。2.4A地图将用于 追踪多肽链并定位侧链的取向 以及辅因的影响。蛋白质的模型将被构建在 MMS-X分子图形系统。为了研究酶促反应 作用机理、各种底物、抑制剂和变构效应物 结合酶并用差分傅立叶方法进行分析。最后， 结构将由Hendrickson-Konnert改进到2.0A分辨率 约束最小二乘法。

The molecular structure of Trimethylamine Dehydrogenase (TMADH) from the methylotrophic bacterium W3A1 will be studied by X-ray diffraction. TMADH is a complex iron-sulfur flavoprotein of 147,000 daltons composed of two non-identical subunits of about 75,000 daltons each. It contains one Fe4S4 cluster and one covalently bound FMN coenzyme per molecule. The enzyme catalyzes the oxidation of trimethylamine to dimethylamine and formaldehyde with subsequent electron transfer to the artificial acceptor phenazine methosulfate. When reduced by substrate the enzyme exhibits very strong spin coupling between the reduced cofactors accompanied by an apparent conformation change. Crystals of TMADH are monoclinic, space group P21 with cell dimensions a=147.63A, b=71.96A, c=83.66A and Beta=97.64A. The crystals diffract to 2.0A resolution. The structure will be analyzed by the isomorphous replacement method first at 6.0A and then at 2.4A resolutions. The 6.0A map will be studied to identify the domain and subunit structure of the enzyme. Native anomalous scattering data will be analyzed to locate the iron-sulful cluster. The 2.4A map will be used to trace the polypeptide chain and locate the orientation of the side chains and of the cofactors. A model of the protein will be constructed on the MMS-X molecular graphics system. In order to study the enzymatic mechanism, various substrates, inhibitors and allosteric effectors will be bound to the enzyme and analyzed by difference Fourier methods. Finally, the structure will be refined to 2.0A resolution by the Hendrickson-Konnert restrained least squares procedure.