基因编码非天然氨基酸研究细胞色素c氧化酶功能

Cytochrome c oxidase (CcO) is a large trans-membrane protein complex.It performs efficient four-electron reduction of oxygen to water, helping to establish a trans-membrane difference of proton electrochemical potential, without releasing toxic, reactive oxygen species (ROS) such as superoxide and peroxide. Because of its importance in biogenetics,fascinating chemistry, and potential applications in fuel cells, it has been intensively investigated by structural biologists, enzymologists, and synthetic chemists. .While tremendous progress has been made, several important questions about the structural features and mechanism still remain. Of particular interest is the unique post-translationally modified covalent cross-link between tyrosine residue and a nearby histidine residue (Tyr-His cross-link) which is confirmed by biochemical and biophysical studies. Importantly, the cross-link is conserved in bacterial and mammal cells, thus implicating a strong evolutionary connection and the functional significance of the cross-link. A major limitation of our understanding is that such a post-translational modification cannot be directly probed through traditional site-directed mutagenesis methods. To contribute to the understanding of the post-translationally modification function, we will make model CcO proteins by engineering an artificial cross-link in target proteins. .Our early studies have found that Mb with encoded 3-imidazolyltyrosine can simulate function of CcO. In this study, a series of 3-imidazolyltyrosine analogues will be further synthesized and site-specific incorporated to specific sites of target proteins by genetic encoding. We will use these new tools to study the catalytic mechanism of CcO. Various means of spectroscopy analysis will be helpful for answering the particularly interesting question of the function of Tyr-His covalent bonding in CcO. Finally, a easy to study model enzyme with similar or higher CcO activity will be obtained. .The study will be helpful for explain the mechanism of CcO and introduce a new tool for protein engineering.

细胞色素c氧化酶(CcO)是一种膜蛋白复合物,它能高效催化氧气转化为水,产生质子梯度,而几乎不产生活性氧。由于其在能量代谢和调节中的重要地位,一直是生物学中重要的研究课题。生化和晶体学研究发现,CcO活性中心存在一个从细菌到哺乳动物细胞中保守的，翻译后修饰形成的 Tyr-His共价交联。由于缺乏有效的对其突变的手段及将这种共价交联引至其他模型蛋白的方法,其确切作用仍是目前研究的焦点。.前期研究发现,通过在容易表达纯化的肌红蛋白中编码含Tyr-His交联元素的咪唑酪氨酸,可以模拟CcO的功能。在此基础上,我们将继续合成一系列功能酪氨酸类似物并将其基因编码到蛋白质中.我们将利用这些新的工具系统研究CcO的催化机理,利用各种光谱学手段进一步揭示Tyr-His共价交联的功能,并获得可以和CcO活性媲美的,易于研究的模拟酶。.该研究将有助于揭示CcO的酶催化机制,并为人工设计新酶提供一个全新的工具。

细胞色素c 氧化酶(CcO)是一种膜蛋白复合物,它能高效催化氧气转化为水,产生质子梯度,而几乎不产生活性氧。由于其在能量代谢和调节中的重要地位,一直是生物学中重要的研究课题。生化和晶体学研究发现,CcO 活性中心存在一个从细菌到哺乳动物细胞中保守的，翻译后修饰形成的 Tyr-His共价交联。由于缺乏有效的对其突变的手段及将这种共价交联引至其他模型蛋白的方法,其确切作用仍是目前研究的焦点。.前期研究发现,通过在容易表达纯化的肌红蛋白中编码含Tyr-His 交联元素的咪唑酪氨酸,可以模拟CcO 的功能。在此基础上,我们继续合成了一系列功能酪氨酸类似物并将其基因编码到蛋白质中.我们利用这些新的工具系统研究了CcO 的催化机理,利用各种光谱学手段进一步揭示了Tyr-His 共价交联的功能,并获得了可以和CcO 活性媲美的,易于研究的模拟酶。.该研究揭示了CcO 的酶催化机制,为人工设计新酶提供了一个全新的工具。