XAS STUDIES OF ACTIVE SITES AND COMPONENT INTERACTIONS IN BACTERIAL MULTICOMPONE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们建议用X射线吸收光谱(XAS)系统地研究一系列细菌多组分单加氧酶(BMMS)中羧酸桥联的非血红素二铁中心的电子结构和几何结构。BMMS由一系列酶组成，能够利用分子氧氧化各种碳氢化合物底物，并需要几个蛋白质组分之间的络合物才能有效催化。我们的目标是阐明双铁活性中心的组分相互作用和结构灵活性在BMMS的催化功能和底物专一性中的作用。所研究的蛋白质体系将包括来自胶囊分枝杆菌(Bath)的可溶性甲烷单加氧酶(SMMO)，来自Stutzeri OX1的甲苯/邻二甲苯单加氧酶(Tomo)，以及来自Pseudomonas Stutzeri OX1的苯酚羟基酶(PH)。我们以前对sMMO和Tomo酶的XAS研究已经提供了羟基酶成分中双铁位置的结构特征，并确定了它在与调节蛋白相互作用时的变化。我们计划扩大XAS实验，以探索这些系统和新型苯酚羟基酶中的其他蛋白质-蛋白质相互作用。具体地说，我们建议：1)获得BMMS羟基酶组分与氧反应形成的中间物种中双铁活性中心的电子和结构信息；2)分析三种羟基酶不同氧化状态下双核铁中心的电子结构和几何结构；3)阐明偶联蛋白对苯酚羟基酶活性中心的影响；以及4)确定羟基酶和含有[2Fe-2S]簇的还原酶组分之间的结合如何影响三种多组份单加氧酶中双金属活性中心和/或[2Fe-2S]中心的电子和计量特性。

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.