Function of the Cofactors of Complex II from Escherichia coli

大肠杆菌复合物 II 辅因子的功能

• 批准号: 9728778
• 负责人: Gary Cecchini
• 金额: $ 27万
• 依托单位: Northern California Institute for Research and Education
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728778&HistoricalAwards=false
• 关键词: Function; Cofactors; Complex; II; Escherichia

Gary Cecchini MCB-9728778 1. Technical Abstract This study is aimed to investigate the function of the cofactors of fumarate reductase (menaquinol-fumarate oxidoreductase) (QFR) and succinate dehydrogenase (succinate-ubiquinone oxidoreductase) (SQR) from Escherichia coli. These two membrane-bound enzyme complexes, found in most prokaryotes and eukaryotes, are structurally and genetically related and are important components of anaerobic and aerobic respiratory chains, respectively. The cofactors of the enzyme include a covalently bound FAD cofactor, bound to the largest membrane extrinsic subunit of the complex which contains the substrate binding site. The iron-sulfur containing subunit of the complex contains three distinct Fe-S clusters. The Fe-S clusters are: Center 1, a 2Fe-2S 2+,1+ cluster; Center 2, a 4Fe-4S 2+,1+ cluster; and Center 3, a 3Fe-4S 1+,0 cluster. The membrane intrinsic domain of the complexes contain two hydrophobic subunits which provide site(s) for the interaction of the enzyme with quinone cofactors. In addition, the SQR complex, (but not QFR) contains a heme prosthetic group. The enzyme complexes can catalyze the same reactions with both SQR and QFR proficient at succinate oxidation, however, only QFR is highly active as a fumarate reductase. The nature of electron transfer through the highly similar enzyme complexes and their cofactors must therefore be responsible for the inability of SQR to efficiently reduce fumarate in catalytic assays. The reasons for this difference will be a major focus of this proposal. Experiments are designed to use biochemical, biophysical, and molecular biological techniques to investigate the protein environment responsible for the functioning of the FAD, Fe-S, and heme cofactors in the enzymes. The enzyme complexes are easily manipulated by site-directed mutagenesis and both wild type and mutant forms of the enzyme can be isolated in very high yield. The large amounts of highly purified enzyme complex which is produced aids in EPR, magnetic circular dichroism (MCD), and resonance Raman spectroscopic methods used to analyze the environment of the Fe-S clusters and the redox potential of these important cofactors. Site-directed mutants of both enzyme complexes will be constructed and analyzed for their effect on the Fe-S clusters. This will be done to determine if the redox potential of the Fe-S clusters can be manipulated in a predictable manner and if the differences in redox potential for the Fe-S clusters of SQR and QFR are the major reason for the differences in the catalytic activity between the two enzyme complexes. Investigations will also be begun to determine if time resolved kinetic measurements of electron transfer through the various cofactors of QFR and SQR can be measured. This will be accomplished by following the photooxidation of pyranine dyes placed specifically at the active site of SQR and QFR and monitoring electron transfer through the FAD, heme, and quinone cofactors of the enzymes. These studies will aid in the understanding of electron transfer reactions through proteins. Important information will also be provided on the differences in electron transfer through the highly similar QFR and SQR enzyme complexes. Gary Cecchini, Ph.D. MCB-9728778 2. Non-technical abstract This study investigates the function of the cofactors of two enzyme complexes important for the function of respiratory function in cells in both an aerobic and anaerobic environment. The enzyme complexes studied are fumarate reductase (menaquinol-fumarate oxidoreductase, QFR) and succinate dehydrogenase (succinate-ubiquinone oxidoreductase, SQR) usually from the bacterium Escherichia coli. These two membrane-bound enzyme complexes, found in most prokaryotes and eukaryotes, are structurally and genetically related. Both enzyme complexes contain a covalently bound FAD cofactor, bound to the largest membrane extrinsic subunit of the complex, which also contains t he dicarboxylate binding site. Each enzyme also contains three distinct iron-sulfur clusters which participate in the electron transfer function of the enzymes. The iron-sulfur clusters have distinct redox potentials spanning a range of several hundred mV. In addition, to the iron-sulfur cluster the SQR complex, but not the QFR complex, contains a heme cofactor as an additional prosthetic group. The major questions being investigated are what factors in the protein environment control the redox potential of the various prosthetic groups in these two enzymes and thus the path of electron flow through the complexes. Under defined conditions succinate dehydrogenase acts as a "tunnel diode" in directing the path of electrons through the various prosthetic groups which act as the electron wires in the protein complex. Using the techniques of site-directed mutagenesis and electron paramagnetic resonance spectroscopy (EPR) the protein environment will be modified to understand how one can alter the rate and directionality of electron flow in these enzymes by modification of the redox potential of the cofactors. These studies will allow a more complete understanding of ways nature designs the micro protein machinery involved in electron transfer reactions. In addition, the studies will show how two very similar enzyme complexes can be poised to catalyze different chemical reactions.

Gary Cecchini MCB-9728778技术摘要本研究旨在研究大肠杆菌中富马酸还原酶（甲基萘酚-富马酸氧化还原酶）（QFR）和琥珀酸脱氢酶（琥珀酸-泛醌氧化还原酶）（SQR）的辅助因子的功能。这两种膜结合酶复合物存在于大多数原核生物和真核生物中，它们在结构和遗传上是相关的，分别是厌氧和有氧呼吸链的重要组成部分。酶的辅因子包括一个共价结合的FAD辅因子，它与含有底物结合位点的复合物的最大膜外亚基结合。该配合物的含铁硫亚基包含三个不同的铁硫团簇。Fe-S簇有：中心1,2Fe-2S 2+，1+簇；中心2、4Fe-4S 2+、1+集群；和中心3，一个3Fe-4S 1+，0集群。复合物的膜内结构域包含两个疏水亚基，为酶与醌辅因子的相互作用提供了位点。此外，SQR复合物（而不是QFR）含有血红素假基。酶复合物可以与精通琥珀酸盐氧化的SQR和QFR催化相同的反应，然而，只有QFR作为富马酸还原酶具有高活性。因此，通过高度相似的酶复合物及其辅助因子的电子转移的性质必然是SQR在催化分析中无法有效还原富马酸的原因。造成这种差异的原因将是本提案的主要焦点。实验旨在使用生化，生物物理和分子生物学技术来研究负责FAD， Fe-S和酶中血红素辅助因子功能的蛋白质环境。酶复合物很容易通过定点诱变操作，并且野生型和突变型的酶都可以以非常高的产量分离出来。产生的大量高纯度酶复合物有助于EPR，磁圆二色性（MCD）和共振拉曼光谱方法用于分析Fe-S簇的环境和这些重要辅因子的氧化还原电位。这两种酶复合物的位点导向突变体将被构建并分析其对Fe-S簇的影响。这样做是为了确定Fe-S簇的氧化还原电位是否可以以可预测的方式进行操纵，以及SQR和QFR的Fe-S簇的氧化还原电位差异是否是两种酶复合物之间催化活性差异的主要原因。研究还将开始确定是否可以测量通过QFR和SQR的各种辅因子的电子转移的时间分辨动力学测量。这将通过跟踪放置在SQR和QFR活性位点的pyranine染料的光氧化并监测通过FAD，血红素和醌辅助因子的电子转移来完成。这些研究将有助于理解通过蛋白质的电子转移反应。还将提供通过高度相似的QFR和SQR酶复合物的电子转移差异的重要信息。Gary Cecchini, Ph.D. MCB-9728778本研究探讨了两种酶复合物的辅助因子在有氧和厌氧环境下对细胞呼吸功能的重要作用。所研究的酶复合物是通常来自大肠杆菌的富马酸还原酶（甲基萘酚-富马酸氧化还原酶，QFR）和琥珀酸脱氢酶（琥珀酸-泛醌氧化还原酶，SQR）。这两种膜结合酶复合物存在于大多数原核生物和真核生物中，在结构和遗传上是相关的。这两种酶复合物都含有一个共价结合的FAD辅助因子，结合到复合物的最大膜外亚基上，该亚基也含有二羧酸结合位点。每种酶还包含三个不同的铁硫簇，它们参与酶的电子传递功能。铁硫团簇具有不同的氧化还原电位，其范围为几百mV。此外，铁硫团簇的SQR配合物（而不是QFR配合物）含有血红素辅助因子作为附加的假基。正在研究的主要问题是蛋白质环境中的哪些因素控制了这两种酶中各种假基的氧化还原电位，从而控制了电子流过复合物的路径。在规定的条件下，琥珀酸脱氢酶作为“隧道二极管”，引导电子通过各种假基的路径，这些假基在蛋白质复合物中充当电子线。利用定点诱变和电子顺磁共振波谱（EPR）技术，蛋白质环境将被修饰，以了解如何通过修饰辅因子的氧化还原电位来改变这些酶中电子流的速率和方向性。这些研究将使我们更全面地了解大自然设计参与电子转移反应的微蛋白质机制的方式。此外，这些研究将展示两种非常相似的酶复合物如何能够催化不同的化学反应。