XAS STUDIES OF ACTIVE SITES AND COMPONENT INTERACTIONS IN BACTERIAL MULTICOMPONE

细菌复合体活性位点和成分相互作用的 XAS 研究

• 批准号: 8169951
• 负责人: KEITH O HODGSON
• 金额: $ 0.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169951
• 关键词: Active Sites; Affect; Bathing; Binding; Catalysis; Characteristics; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; Dioxygen; Electronics; Enzymes; Family; Funding; Goals; Grant; Hydrocarbons; Institution; Iron; Methane hydroxylase; Mixed Function Oxygenases; Molecular; Oxidoreductase; Oxygen; Phenol 2-monooxygenase; Proteins; Pseudomonas stutzeri; Reaction; Research; Research Personnel; Resources; Role; Series; Site; Source; Spectrum Analysis; Structure; Substrate Specificity; System; United States National Institutes of Health; absorption; carboxylate; flexibility; genetic regulatory protein; novel; oxidation; protein protein interaction; research study; toluene 2-xylene monooxygenase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to probe systematically the electronic and geometric structure of the carboxylate-bridged nonheme diiron sites in a series of bacterial multicomponent monooxygenases (BMMs) using x-ray absorption spectroscopy (XAS). BMMs comprise a family of enzymes capable of utilizing molecular oxygen to oxidize a variety of hydrocarbon substrates, and require complexes between several protein components for efficient catalysis. Our goal is to elucidate the role of component interactions and structural flexibility at the diiron active center in the catalytic function and substrate specificity of BMMs. The proteins systems studied will include soluble methane monooxygenase (sMMO) from M. capsulatus (Bath), toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. Our previous XAS studies of the sMMO and ToMO enzymes have afforded a structural characterization of the diiron site in the hydroxylase component, and identified its changes upon interaction with the regulatory proteins. We plan to extend XAS experiments to explore other protein-protein interactions in these systems and in the novel phenol hydroxylase. Specifically, we propose i) to obtain electronic and structural information on the diiron active center in intermediate species formed in the reaction of the hydroxylase component of BMMs with dioxygen; ii) to analyze the electronic and geometric structure of the dinuclear iron site in different oxidation states in the three hydroxylases; iii) to elucidate the effect of the coupling protein on the active center in the phenol hydroxylase; and iv) to determine how the binding between the hydroxylase and reductase component containing the [2Fe-2S] cluster affects the electronic and metrical characteristics of the dimetal active site and/or [2Fe-2S] center in the three multicomponent monooxygenases.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们建议使用 X 射线吸收光谱 (XAS) 系统地探讨一系列细菌多组分单加氧酶 (BMM) 中羧酸桥非血红素二铁位点的电子和几何结构。 BMM 包含一系列能够利用分子氧氧化各种碳氢化合物底物的酶，并且需要几种蛋白质成分之间的复合物才能有效催化。我们的目标是阐明二铁活性中心的组分相互作用和结构灵活性在 BMM 的催化功能和底物特异性中的作用。研究的蛋白质系统将包括来自 M. capsulatus (Bath) 的可溶性甲烷单加氧酶 (sMMO)、来自施氏假单胞菌 OX1 的甲苯/邻二甲苯单加氧酶 (ToMO) 和来自施氏假单胞菌 OX1 的苯酚羟化酶 (PH)。我们之前对 sMMO 和 ToMO 酶的 XAS 研究提供了羟化酶组分中二铁位点的结构特征，并确定了其在与调节蛋白相互作用时的变化。我们计划扩展 XAS 实验，以探索这些系统和新型酚羟化酶中的其他蛋白质-蛋白质相互作用。具体来说，我们建议 i) 获得 BMM 羟化酶成分与双氧反应中形成的中间物质中二铁活性中心的电子和结构信息； ii) 分析三种羟化酶中不同氧化态的双核铁位点的电子和几何结构； iii) 阐明偶联蛋白对苯酚羟化酶活性中心的影响； iv) 确定羟化酶和含有[2Fe-2S]簇的还原酶组分之间的结合如何影响三种多组分单加氧酶中双金属活性位点和/或[2Fe-2S]中心的电子和计量特征。