Function of the Cofactors of Complex II from Escherichia coli

加里·塞奇尼MCB-9728778 1.技术摘要本研究旨在探讨大肠埃希菌富马酸还原酶(富马酸氧化还原酶)和琥珀酸脱氢酶(琥珀酸辅酶Q氧化还原酶)辅酶因子的功能。这两种膜结合酶复合体存在于大多数原核生物和真核生物中，它们在结构和基因上是相关的，分别是厌氧呼吸链和好氧呼吸链的重要组成部分。该酶的辅因子包括共价结合的FAD辅因子，该辅因子结合到含有底物结合部位的复合体的最大膜外源亚基上。该络合物的铁硫亚基含有三个不同的铁-S簇合物。Fe-S团簇是：中心1，2Fe-2S2+，1+团簇；中心2，4Fe-4S2+，1+团簇；中心3，3Fe-4S1+，0团簇。该复合体的膜固有结构域含有两个疏水亚基，它们为酶与苯醌辅助因子的相互作用提供了位点(S)。此外，SQR复合体(但不是QFR)含有一个血红素假体基团。这些酶复合体可以催化与SQR和QFR同样擅长琥珀酸氧化的反应，但只有QFR作为富马酸还原酶具有很高的活性。因此，通过高度相似的酶复合体及其辅因子进行的电子转移的性质必然是SQR在催化分析中不能有效地还原富马酸的原因。这一差异的原因将是这项提案的主要焦点。实验旨在利用生化、生物物理和分子生物学技术来研究蛋白质环境，这些环境对酶中FAD、铁-S和血红素辅助因子的功能负责。酶复合体很容易通过定点突变进行操作，并且可以非常高的产率分离野生型和突变型的酶。产生的大量高纯度的酶复合体有助于电子顺磁共振、磁圆二色谱和共振拉曼光谱方法分析Fe-S团簇的环境和这些重要辅因子的氧化还原电位。将构建这两种酶复合体的定点突变体，并分析它们对Fe-S簇的影响。这将被用来确定Fe-S团簇的氧化还原电位是否能够以可预测的方式被操纵，以及SQR和QFR的Fe-S团簇的氧化还原电位的差异是否是导致两种酶络合物催化活性差异的主要原因。还将开始调查，以确定是否可以测量通过QFR和SQR的各种辅助因子进行的电子转移的时间分辨动力学测量。这将通过跟踪特定放置在SQR和QFR活性位置的吡喃染料的光氧化并通过酶的FAD、血红素和苯醌辅助因子监测电子转移来实现。这些研究将有助于理解蛋白质中的电子转移反应。还将提供有关通过高度相似的QFR和SQR酶复合体进行电子转移的差异的重要信息。加里·切奇尼博士，MCB-9728778 2.非技术摘要本研究探讨了在有氧和无氧环境中对细胞呼吸功能起重要作用的两种酶复合体的辅因子的功能。所研究的酶复合体是富马酸还原酶(MEQR)和琥珀酸脱氢酶(SQR)，这两种酶通常来自大肠杆菌。这两种膜结合的酶复合体存在于大多数原核生物和真核生物中，在结构和基因上是相关的。两种酶复合体都含有共价结合的FAD辅因子，结合到复合体最大的膜外源亚基上，该亚基还含有二元酸结合部位。每种酶还包含三个不同的铁-硫簇，它们参与酶的电子转移功能。铁-硫团簇在几百mV的范围内具有明显的氧化还原电位。此外，对于铁-硫簇，SQR复合体，而不是QFR复合体，包含一个血红素辅因子作为额外的修复基团。正在研究的主要问题是，蛋白质环境中的哪些因素控制着这两种酶中不同辅基的氧化还原电位，从而控制电子通过复合体的路径。在特定的条件下，琥珀酸脱氢酶起到“隧道二极管”的作用，引导电子穿过蛋白质复合体中充当电子线的各种假体基团。利用定点突变和电子顺磁共振波谱(EPR)技术，蛋白质环境将被改变，以了解人们如何通过改变辅因子的氧化还原电位来改变这些酶中电子流动的速度和方向性。这些研究将使人们能够更全面地了解大自然设计参与电子转移反应的微观蛋白质机制的方式。此外，这些研究将展示两个非常相似的酶复合体如何能够催化不同的化学反应。

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.