MauG-preMADH intermediate structures: Insight into long range electron transfer

MauG-preMADH 中间体结构：深入了解长程电子转移

• 批准号: 8266014
• 负责人: Erik T Yukl
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8266014
• 关键词: Aging; Anabolism; Binding; Biological; Cell Respiration; Chemistry; Complex; Crystallography; Data Collection; Disease; Dissociation; Electron Transport; Electrons; Enzymes; Free Radicals; Freezing; Goals; Health; Heme; Human; Hydrogen Peroxide; Hydroxyl Radical; Incubated; Ligands; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Modification; Mole the mammal; Molecular; Mutation; Nature; Oxidants; Oxygen; Pathway interactions; Post-Translational Protein Processing; Process; Property; Proteins; Proteolysis; Protons; Quinones; Reaction; Reactive Oxygen Species; Resolution; Roentgen Rays; Sampling; Series; Site; Solutions; Spectrum Analysis; Structure; System; Techniques; Time; Tryptophan; Variant; X-Ray Crystallography; cell injury; cofactor; crosslink; insight; interest; methylamine dehydrogenase; oxidation; prevent; protein aminoacid sequence; research study; tandem mass spectrometry

DESCRIPTION (provided by applicant): An unusual di-heme enzyme MauG catalyzes the 6-electron oxidation of a precursor methylamine dehydrogenase with a monohydroxylated 2Trp57 (preMADH) to form the mature tryptophan tryptophyl quinone (TTQ) cofactor. The reaction proceeds via three, two-electron oxidations involving the insertion of the second oxygen atom into 2Trp57, formation of the crosslink between 2Trp57 and 2Trp108, and oxidation to the quinone. The order of these modifications is unknown. MauG can use 3 moles of either H2O2 or O2 plus reducing equivalents to oxidize preMADH. Addition of stoichiometric H2O2 to MauG results in the formation of a catalytically competent bis-Fe(IV) species with one of the hemes in a Fe(IV)=O state while the other is a Fe(IV) species ligated by His and Tyr ligands. This intermediate demonstrates an unprecedented method for stabilizing a highly oxidizing species equivalent to an Fe(V). A crystal structure of the preMADH- MauG complex shows that the site of oxygen binding and activation is over 30 z from the TTQ site. Addition of excess H2O2 to the crystals results in formation of TTQ, demonstrating that the crystals are catalytically active, that each step occurs processively without dissociation of the complex, and that oxidation occurs via long range inter-protein electron transfer. One goal of this project is to structurally characterize the bis-Fe(IV) catalytic intermediate of MauG. The other objective is to characterize the order and nature of each of the 2-electron oxidation reactions occurring at the TTQ site. These biosynthetic intermediates will be generated in crystallo and structurally characterized. Mass spectrometry will also be used to confirm the results of crystallography experiments and to characterize steps involving proton transfer or radical formation, which has been implicated in the first oxidation step. These experiments promise to provide significant insight into methods of radical and high- valent oxidant stabilization within proteins as well as mechanisms of inter-protein electron transfer. These processes underpin aerobic metabolism and have been implicated in various disease states and aging, making them significant to human health.

描述（由申请人提供）：一种不寻常的二血红素酶 MauG 催化前体甲胺脱氢酶与单羟基化 2Trp57 (preMADH) 的 6 电子氧化，形成成熟的色氨酸色氨酸醌 (TTQ) 辅因子。该反应通过三个双电子氧化进行，包括将第二个氧原子插入 2Trp57、2Trp57 和 2Trp108 之间形成交联，以及氧化成醌。这些修改的顺序未知。 MauG 可以使用 3 摩尔的 H2O2 或 O2 加上还原当量来氧化 preMADH。将化学计量的 H2O2 添加到 MauG 中会形成具有催化能力的双 Fe(IV) 物质，其中一个血红素处于 Fe(IV)=O 状态，而另一个是通过 His 和 Tyr 配体连接的 Fe(IV) 物质。该中间体展示了一种前所未有的方法来稳定相当于 Fe(V) 的高氧化性物质。 preMADH-MauG 复合物的晶体结构显示氧结合和活化位点距离 TTQ 位点超过 30 z。向晶体中添加过量的 H2O2 会导致 TTQ 的形成，这表明晶体具有催化活性，每个步骤都在不解离的情况下进行，并且氧化是通过长程蛋白质间电子转移发生的。该项目的目标之一是对 MauG 的双 Fe(IV) 催化中间体进行结构表征。另一个目标是表征 TTQ 位点发生的每个 2 电子氧化反应的顺序和性质。这些生物合成中间体将以晶体形式产生并进行结构表征。质谱分析还将用于确认晶体学实验的结果，并表征涉及质子转移或自由基形成的步骤，这与第一个氧化步骤有关。这些实验有望为蛋白质内自由基和高价氧化剂稳定的方法以及蛋白质间电子转移的机制提供重要的见解。这些过程支撑有氧代谢，并与各种疾病状态和衰老有关，对人类健康具有重要意义。