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Cytochrome c oxidase: structure, function and malfunction

细胞色素 C 氧化酶：结构、功能和故障

基本信息

批准号：
MR/M00936X/1
负责人：
Amandine MARECHAL
金额：
$ 131.49万
依托单位：
Birkbeck, University of London
依托单位国家：
英国
项目类别：
Fellowship
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FM00936X%2F1
关键词：
Cytochrome oxidase structure function malfunction

项目摘要

To live we need a permanent supply of energy. This is provided to our cells by a cascade of reactions that breaks down the food we eat into a universal fuel: the ATP. This process mainly occurs in organelles called mitochondria and is known as cellular respiration. The main machinery that mitochondria use to produce ATP is the respiratory chain. It is composed of four complexes, embedded in the mitochondrial inner membrane, that work together to build up an electrochemical gradient called the proton motive force and which drives ATP synthesis. Most of this gradient is in the form of protons which are pumped across the inner mitochondrial membrane by the respiratory chain complexes.An increasing number of human pathologies are associated with defects in components of the respiratory chain. In many instances, this is because the malfunction has a direct impact on their primary role in energy production via the proton gradient that they form, or because it leads to an increased production of damaging free radicals. Cytochrome c oxidase (CcO) is the terminal enzyme of our respiratory chain. It transforms the oxygen we breathe into water and greatly contributes to the generation of the proton gradient. Alterations (or mutations) in its structure have been linked with diverse pathologies such as myopathy, therapy-resistant epilepsy, neurological diseases and prostate cancer.Although the overall chemistry of mitochondrial CcO is fairly well understood, it has proven much more difficult to determine how this produces the essential proton gradient. Various hypotheses were formulated based on the available structures of the enzyme (of which only one is of mitochondrial origin, in this case bovine), but were challenged by mutagenesis work performed on smaller bacterial homologues. Today it appears that the major drawback in understanding the mechanism of mitochondrial CcO, and the effects of human disease-related mutations in particular, is the lack of a system to generate large amounts of purified protein containing defined point mutations.Remarkably, the CcO that is present in Baker's yeast mitochondria is almost identical to that in human mitochondria. The nuclear and mitochondrial DNAs which encode CcO are both amenable to mutagenesis so alterations can be made in any part of the CcO structure to investigate its function. We have thus engineered a yeast system to allow large-scale production of mutants and will use it to address fundamental questions relative to human mitochondrial CcOs.At first, we will identify the route taken by the protons to cross the protein structure by measuring CcO's ability to pump protons after alterations have been made in chosen area. We will then use advanced techniques like infrared spectroscopy to look at the concerted movement of atoms within CcO's structure and bring experimental evidences of the mechanism following which protons are being pumped. This should tell us more about the principles that govern and control the complex activity and will be our starting point to investigate how factors or signals external to the reaction centre can, in vivo, regulate CcO's activity. This will be of particular interest to understand how the human CcO has adapted to different energy requirements depending on tissue type. We will aim to obtain a detailed 3D structure of the yeast CcO to confirm our hypotheses. As we unravel the details of CcO's action, we will introduce identified human disease-related mutations in our yeast system in order to investigate the nature of their malfunction. Finally, we will aim at progressively incorporating the human genes or parts of the human enzyme in our yeast system. This will create as even better model for the study of human diseases and the development and testing of new therapies.

为了生存，我们需要永久的能源供应。这是通过一系列反应提供给我们的细胞的，这些反应将我们所吃的食物分解成一种通用的燃料：三磷酸腺苷。这一过程主要发生在称为线粒体的细胞器中，被称为细胞呼吸。线粒体用来产生三磷酸腺苷的主要机械是呼吸链。它由嵌入线粒体内膜的四个复合体组成，它们共同作用建立一个称为质子动力的电化学梯度，并驱动ATP的合成。这种梯度大部分是以质子的形式存在的，这些质子被呼吸链复合体泵过线粒体内膜。越来越多的人类疾病与呼吸链成分的缺陷有关。在许多情况下，这是因为故障通过它们形成的质子梯度直接影响它们在能量生产中的主要作用，或者因为它导致破坏性自由基的产生增加。细胞色素c氧化酶(CcO)是呼吸链的末端酶。它将我们呼吸的氧气转化为水，并极大地促进了质子梯度的产生。其结构的改变(或突变)与多种病理有关，如肌病、难治性癫痫、神经系统疾病和前列腺癌。尽管线粒体CcO的整体化学已被很好地了解，但事实证明，要确定这是如何产生必要的质子梯度要困难得多。基于酶的现有结构(其中只有一种是线粒体起源的，在这种情况下是牛的)，提出了各种假说，但受到了对较小细菌同源物进行的突变工作的挑战。今天，在理解线粒体CcO的机制，特别是人类疾病相关突变的影响方面，主要的缺陷似乎是缺乏一个系统来产生大量含有明确点突变的纯化蛋白。值得注意的是，存在于面包师酵母线粒体中的CcO与人类线粒体中的CcO几乎完全相同。编码CcO的核和线粒体DNA都容易发生突变，因此可以对CcO结构的任何部分进行改变，以研究其功能。因此，我们设计了一个酵母系统，允许大规模生产突变体，并将使用它来解决与人类线粒体CCOS相关的基本问题。首先，我们将通过测量CCOO在选定区域发生改变后泵送质子的能力来确定质子穿过蛋白质结构的路径。然后，我们将使用先进的技术，如红外光谱，来观察CCOO结构中原子的协调运动，并提供质子被泵浦的机制的实验证据。这应该会让我们更多地了解支配和控制复杂活动的原理，并将成为我们研究反应中心外部因素或信号如何在体内调节CcO活动的起点。这对了解人类CcO如何适应不同组织类型的不同能量需求将特别感兴趣。我们将致力于获得酵母CcO的详细3D结构来证实我们的假设。随着我们揭开Cco行动的细节，我们将在我们的酵母系统中引入已识别的人类疾病相关突变，以调查它们故障的性质。最后，我们的目标是逐步将人类基因或部分人类酶整合到我们的酵母系统中。这将为人类疾病的研究以及新疗法的开发和测试创造更好的模式。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy.

DOI：
10.1016/j.bbabio.2018.05.018
发表时间：
2018-09
期刊：
Biochimica et biophysica acta. Bioenergetics
影响因子：
0
作者：
Maréchal A;Hartley AM;Warelow TP;Meunier B;Rich PR
通讯作者：
Rich PR

Spontaneous assembly of redox-active iron-sulfur clusters at low concentrations of cysteine.

DOI：
10.1038/s41467-021-26158-2
发表时间：
2021-10-11
期刊：
Nature communications
影响因子：
16.6
作者：
Jordan SF;Ioannou I;Rammu H;Halpern A;Bogart LK;Ahn M;Vasiliadou R;Christodoulou J;Maréchal A;Lane N
通讯作者：
Lane N

Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1