Reaction intermediates in the O2 activation mechanism of an extradiol dioxygenase: visualising catalytic protons and active site dynamics in crystallo

氧是地球上生命的基础，但它是一种令人惊讶的非反应性分子，必须先被激活，然后才能被细胞利用。对于生命科学研究人员来说，了解氧气是如何被激活的是一个关键问题。氧气的激活是神秘的，为了人类的健康，必须适当地控制它。如果这一过程失控，活性氧会释放到细胞内，造成与心脏病和衰老有关的损害。在这个项目中，我们正在研究一种酶，一种加速细胞内化学反应的生物分子，是如何激活氧气的。我们的酶使用活性氧来分解一系列化学物质，其中包括几种天然和人造污染物。了解这种酶的工作原理不仅有助于解释氧激活是如何控制的，还可能有助于开发“绿色化学”来清理污染。我们打算通过用强X射线或中子束轰击这种酶来检测这种酶，这种技术被称为结晶学。这种方法使我们能够在原子水平上看到事件，所以我们可以直接可视化氧是如何与酶的活性中心相互作用的。令人兴奋的是，将酶冷却到-180摄氏度会阻止化学反应，这样我们就可以看到不同阶段的反应。将结晶学与其他技术相结合，如突变，我们可以改变酶的活性中心，我们可以开始了解控制氧激活和随后的化学作用的潜在化学和物理过程。我们还将使用计算机建模来识别我们目前在实验室中无法观察到的化学特征。这些计算机模型将首先根据我们已知的实验数据进行验证，以确保结果匹配。然后，它们可以用来预测在现实生活中非常困难或不可能做的实验的结果。我们希望通过使用这种令人兴奋的尖端技术组合，我们可以更完整地理解涉及氧激活的化学是如何被生物控制的。

Oxygen is fundamental to life on earth, yet it is a surprisingly unreactive molecule that must be activated before it can be used by cells. Understanding how oxygen is activated is a critical question for life science researchers. Oxygen activation is mysterious and must be properly controlled for good health in humans. If the process happens uncontrollably, reactive oxygen species can be released into cells, causing damage linked to heart disease and ageing. In this project we are studying how an enzyme, a biological molecule that accelerates chemical reactions in the cell, activates oxygen. Our enzyme uses the activated oxygen to breakdown a family of chemicals that includes several natural and man-made pollutants. Understanding how this enzyme works will not only help explain how oxygen activation is controlled, but may also help in developing 'green chemistry' to clean up pollution. We intend examine this enzyme by bombarding it with intense X-ray light or a neutron beam in a technique known as crystallography. This method allows us to see events on the atomic level, so we can directly visualise how the oxygen interacts with the active centre of the enzyme. Excitingly, cooling the enzyme to -180C arrests the chemical reaction so that we can visualise the reaction at different stages. Combining crystallography with other techniques, like mutagenesis, where we alter the enzyme active centre, we can begin to understand the underlying chemical and physical processes that control oxygen activation and the ensuing chemistry. We will also employ computer modelling to discern chemical features we cannot currently observe in the lab. These computer models will first be validated against our known experimental data to ensure the results match. Then, they can be used to predict the results of experiments that are very difficult or impossible to do in real-life. We hope that by using this exciting combination of cutting-edge techniques we can achieve a more complete understanding of how chemistry involving oxygen activation is controlled by biology.