STRUCTURAL STUDIES OF COMPLEX IRON SULFUR FLAVOPROTEINS

描述（改编自申请人的摘要）：两种化合物的结构研究 将携带参与胺氧化的氧化还原蛋白系统 出去 其中一个系统由铁硫黄素蛋白组成， 三甲基胺脱氢酶（TMADH），其经历分子内 电子从黄素转移到铁硫中心， 催化，和电子转移黄素蛋白（ETF），特定的 TMADH的电子受体。 另一个系统，酵母铜胺氧化酶 (YAO)是一种醌蛋白。 它含有托蒽醌（TPQ），一种催化剂， 一种由基因编码的酪氨酸侧链和铜原子衍生的辅因子。 TMADH的结构已知为1.7 A分辨率，并且几种突变体具有 准备和鉴定。 这些研究中提出的问题 是FMN共价键的形成和作用，特异性和 底物结合在活性位点的稳定化， 底物氧化，分子内电子转移动力学控制 以及分子间电子转移步骤的性质。 的 这些突变体中的几个的结构将被确定，并且另外的 将设计突变体并对其进行结构表征。 天然 结构的分辨率将从快速冷冻晶体扩展到1.0 A 使用同步辐射和几个功能的结构， 衍生物，旨在分子内电子转移步骤，将是 完成 ETF的结构将完成，并用于模拟 与TMADH形成分子间电子转移复合物。 最近通过分子置换法确定了YAO的结构 和单一同晶置换。 精细结构显示 TPQ的取向和铜中心的活性配置， 休眠酶 几种药的功能衍生物的结构 和几个突变体的设计，以探测其生物和催化 属性将被确定。 这些研究所涉及的问题是 TPQ的生物合成，TPQ附近的某些保守残基的作用 TPQ和其他地方，底物特异性，结构和功能，在 活性位点和活性位点对底物的接近。 该结构将 也可用于设计另外的突变体， 分析了

DESCRIPTION (Adapted from applicant's abstract): Structural studies of two redox protein systems involved in the oxidation of amines will be carried out. One of the systems consists of an iron-sulfur flavoprotein, trimethylamine dehydrogenase (TMADH), which undergoes intramolecular electron transfer from the flavin to the iron-sulfur center during catalysis, and an electron transferring flavoprotein (ETF), the specific the electron acceptor for TMADH. The other system, yeast copper amine oxidase (YAO), is a quinoprotein. It contains topaquinone (TPQ), a catalytic cofactor derived from a gene-encoded tyrosine side chain and a copper atom. The structure of TMADH is known at 1.7 A resolution and several mutants have been prepared and characterized. The questions addressed in these studies are the formation and role of the covalent link to FMN, the specificity and stabilization of substrate binding at the active site, the mechanism of substrate oxidation, control of intramolecular electron transfer kinetics and the nature of the intermolecular electron transfer step to ETF. The structures of several of these mutants will be determined and additional mutants will be designed and structurally characterized. The native structure will be extended in resolution to 1.0 A from flash frozen crystals using synchrotron radiation and the structures of several functional derivatives, aimed at the intramolecular electron transfer step, will be completed. The structure of ETF will be completed and used to model the intermolecular electron transfer complex with TMADH. The structure of YAO has recently been determined by molecular replacement and single isomorphous replacement. The refined structure shows the orientation of TPQ and of the copper center in the active configuration of the resting enzyme. The structures of several functional derivatives of YAO and of several mutants designed to probe its biogenic and catalytic properties will be determined. The questions addressed by these studies are the biosynthesis of TPQ, the roles of certain conserved residues near the TPQ and elsewhere, substrate specificity, structure and function at the active site, and access of the active site to substrate. The structure will also be used to design additional mutants which will also be structurally analyzed.