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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们建议探测系统的电子和几何结构的羧酸根桥接的nonheme二铁网站在一系列的细菌多组分单加氧酶（BDEs）使用X-射线吸收光谱（XAS）。生物酶包括能够利用分子氧来氧化多种烃底物的酶家族，并且需要几种蛋白质组分之间的复合物来进行有效催化。我们的目标是阐明组分相互作用和结构灵活性的作用在二铁活性中心的催化功能和底物特异性的Bcl 3。所研究的蛋白质系统将包括来自M.荚膜梭菌（Bath）的甲苯/邻二甲苯单加氧酶（ToMO）、来自施氏假单胞菌OX 1的甲苯/邻二甲苯单加氧酶（ToMO）和来自施氏假单胞菌OX 1的苯酚羟化酶（PH）。我们以前的XAS研究的sMMO和ToMO酶提供了一个结构表征的羟化酶组分中的二铁网站，并确定其变化后，与调节蛋白的相互作用。我们计划扩展XAS实验，以探索在这些系统和新的苯酚羟化酶中的其他蛋白质-蛋白质相互作用。具体地说，我们提出：（1）获得BH 3羟化酶组分与分子氧反应形成的中间体中二铁活性中心的电子和结构信息，（2）分析三种羟化酶中不同氧化态双核铁中心的电子和几何结构，（3）阐明偶联蛋白对苯酚羟化酶活性中心的影响，（4）研究偶联蛋白对苯酚羟化酶活性中心的影响，（5）研究偶联蛋白对苯酚羟化酶活性中心的影响，（6）研究偶联蛋白对苯酚羟化酶活性中心的影响，（7）研究偶联蛋白对苯酚羟化酶活性中心的影响。和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]中心的电子和测量特性。

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.