Cytochrome c oxidase: structure, function and malfunction

细胞色素 C 氧化酶：结构、功能和故障

• 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

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.

要活下去，我们需要永久的能源供应。这是通过一系列反应将我们食用为通用燃料的反应提供给我们细胞的：ATP。该过程主要发生在称为线粒体的细胞器中，被称为细胞呼吸。线粒体用于产生ATP的主要机械是呼吸链。它由四个络合物组成，嵌入了线粒体内膜中，它们共同构建一种称为质子动力的电化学梯度，并驱动ATP合成。这种梯度的大部分都是质子的形式，这些质子是通过呼吸链复合物在内部线粒体膜上泵送的。越来越多的人类病理与呼吸链成分缺陷有关。在许多情况下，这是因为故障通过质子梯度形成的质子梯度在能源生产中的主要作用有直接影响，或者导致损害自由基的产生增加。细胞色素C氧化酶（CCO）是我们呼吸链的末端酶。它将我们呼吸到水中的氧气转化，并极大地有助于产生质子梯度。其结构中的改变（或突变）与多种病理有关，例如肌病，耐药性癫痫，神经系统疾病和前列腺癌。尽管很好地理解了线粒体CCO的整体化学，但事实证明，它很难确定如何产生基本的蛋白质梯度。根据酶的可用结构（其中只有一个是线粒体起源，在这种情况下为牛）制定了各种假设，但受到对较小细菌同源物进行的诱变工作的挑战。如今，看来理解线粒体CCO机理的主要缺点，尤其是与人类疾病相关的突变的作用，是缺乏产生大量纯化蛋白质含有定义点突变的系统的系统。可以明显地存在于Baker的Yeast Mitochoconiria中与人类Mitochondria中几乎相同的CCO。编码CCO的核和线粒体DNA都可以诱变，因此可以在CCO结构的任何部分进行改变以研究其功能。因此，我们已经设计了一个酵母系统来允许大规模生产突变体，并将使用它来解决相对于人类线粒体CCOS的基本问题。首先，我们将确定质子通过在所选区域中衡量CCO泵送质子的能力，通过测量CCO泵送蛋白质结构的能力。然后，我们将使用红外光谱等先进技术来研究原子在CCO结构中的一致运动，并带来泵送质子的机制的实验证据。这应该告诉我们更多有关管理和控制复杂活动的原则的信息，并将成为我们的起点，以调查反应中心外部的因素或信号如何在体内调节CCO的活动。了解人类CCO如何根据组织类型适应不同的能量需求。我们将旨在获得酵母CCO的详细3D结构，以确认我们的假设。当我们揭示CCO作用的细节时，我们将在酵母系统中引入已确定的与人类疾病相关的突变，以研究其故障的性质。最后，我们将旨在将人类基因或人类酶的部分纳入我们的酵母菌系统。这将成为研究人类疾病以及新疗法的发展和测试的更好模型。

项目成果

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
• 作者: Maréchal A;Hartley AM;Warelow TP;Meunier B;Rich PR
• 通讯作者: Rich PR

Cryo-EM structure of a monomeric yeast S. cerevisiae complex IV isolated with maltosides: Implications in supercomplex formation.

• DOI: 10.1016/j.bbabio.2022.148591
• 发表时间: 2022-07
• 期刊: Biochimica et biophysica acta. Bioenergetics
• 作者: Gabriel Ing;Andrew M. Hartley;N. Pinotsis;A. Maréchal

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

• DOI: 10.1038/s41594-018-0172-z
• 发表时间: 2019-01-01
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Hartley, Andrew M.;Lukoyanova, Natalya;Marechal, Amandine
• 通讯作者: Marechal, Amandine