Cytochrome c oxidase: structure, function and malfunction

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，这些反应将我们吃的食物分解成一种通用的燃料：ATP。这一过程主要发生在称为线粒体的细胞器中，称为细胞呼吸。线粒体用来产生ATP的主要机制是呼吸链。它由嵌入线粒体内膜的四种复合物组成，它们共同作用以建立称为质子动力的电化学梯度，并驱动ATP合成。这种梯度的大部分是以质子的形式，通过呼吸链复合体泵送穿过线粒体内膜。越来越多的人类病理与呼吸链组分的缺陷有关。在许多情况下，这是因为故障直接影响它们通过形成质子梯度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。线粒体CcO结构的改变（或突变）与多种病理学有关，如肌病、难治性癫痫、神经系统疾病和前列腺癌。虽然线粒体CcO的整体化学性质已经相当清楚，但要确定它如何产生必要的质子梯度要困难得多。基于酶的可用结构制定了各种假设（其中只有一种是线粒体来源的，在这种情况下是牛），但受到较小的细菌同源物上进行的诱变工作的挑战。目前看来，理解线粒体CcO机制的主要缺陷，特别是人类疾病相关突变的影响，是缺乏一个系统来产生大量的纯化蛋白质含有定义的点突变。值得注意的是，存在于面包酵母线粒体中的CcO几乎与人类线粒体中的相同。编码CcO的核DNA和线粒体DNA都易于诱变，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，允许大规模生产的突变体，并将使用它来解决基本的问题，相对于人类线粒体CcO。首先，我们将确定的路线所采取的质子穿过蛋白质结构，通过测量CcO的能力，泵质子后，已在选定的区域进行了更改。然后，我们将使用红外光谱等先进技术来观察CcO结构中原子的协同运动，并提供质子泵送机制的实验证据。这应该告诉我们更多的原则，管理和控制复杂的活动，并将是我们的出发点，调查如何因素或信号外部的反应中心，在体内，调节CcO的活动。这对于理解人类CcO如何适应取决于组织类型的不同能量需求将是特别感兴趣的。我们的目标是获得酵母CcO的详细3D结构，以证实我们的假设。当我们解开CcO作用的细节时，我们将在我们的酵母系统中引入已鉴定的人类疾病相关突变，以研究其故障的性质。最后，我们的目标是逐步将人类基因或人类酶的一部分纳入我们的酵母系统。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型。

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.