The exact chemical identity of reactive intermediates in O2-dependent uric acid biodegradation

与大多数其他动物不同，人类可以处理体内大量的尿酸。这是因为在进化过程中，我们逐渐沉默了一个负责产生一种叫做尿酸氧化酶（UOX）的基因。这种酶能够将尿酸分解成更易溶解的化合物。人类适应高尿酸水平的原因尚不完全清楚，有趣的是，缺乏功能性UOX酶的小鼠在出生后不久就会死亡。在某些导致尿酸进一步升高的病理条件下，患者服用UOX以帮助恢复正常水平。结晶尿酸沉积也是痛风疾病的标志。UOX需要分子氧（O2）来完成分解尿酸的任务。O2是一种非常有趣的分子，因为在正常的“静息”状态下（空气中的形式），由于与它的电子结构有关的原因，它不想与绝大多数有机分子发生反应。氧需要活化才能反应。然而，一个主要的问题是，一旦“活化”的氧气可以不加选择地与许多生物分子发生反应，造成有害的后果。例如，活性氧（ROS）是“活性氧”的破坏形式，在衰老中起着重要作用。因此，除了“活性氧”的产生外，氧生物化学的另一个挑战是它的控制。在这项工作中，我们将研究UOX如何利用氧气来分解尿酸。有趣的是，UOX属于一小群酶，它们可以将氧气带入与其有机底物的反应中，并在有限的化学工具的支配下将反应导向所需的产物。事实上，由于氧气活化不是一件容易的事，绝大多数酶依赖于特殊的额外成分，如金属和/或有机辅助因子来形成“活性氧”。UOX不需要这些额外的帮助程序，因此理解它是如何工作的特别有趣。使用一种叫做x射线晶体学的技术，它可以以非常高的分辨率“看到”小到尿酸氧化酶（比人类头发的厚度小1万倍）的分子的3D结构，我们已经能够可视化尿酸降解过程中酶的快照（反应中间体），包括O2被困在底物上方的状态。这些快照使我们对尿酸氧化酶的工作原理提出了一些假设。我们现在处于一个极好的位置，可以研究UOX化学中最难以捉摸和最重要的性质。为此，我们将使用一种称为中子晶体学的技术，它可以探测到即使用x射线晶体学也无法观察到的原子（氢）。通过结合中子晶体学、x射线晶体学、现代光谱学技术和先进的量子力学理论方法来探测实验无法获得的状态，我们将了解在UOX功能背景下O2生物化学的一般规律。这种综合方法不仅可以加深对UOX的了解，还可以加深对氧气的了解，氧气是地球上生命的重要组成部分。

Differently from the majority of other animals humans cope with large quantities of uric acid in their bodies. This is because during evolution we have progressively silenced a gene responsible for the production of an enzyme called urate oxidase (UOX). This enzyme is able to break down uric acid into more soluble compounds. The reasons for human adaptation to high uric acid levels are not entirely clear and, interestingly, mice without a functional UOX enzyme die shortly after birth. Under certain pathological conditions that cause a further increase in uric acid UOX is administered to patients to help restore normal levels. Crystalline uric acid deposits are also the hallmark of gout disease.UOX requires molecular oxygen (O2) to perform its task of breaking down uric acid. O2 is a very interesting molecule as in its normal "resting" state (the form present in the air) does not want to react with the vast majority of organic molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once "activated" oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of "active oxygen" that play an important role in aging. Therefore, besides the generation of "active oxygen", another challenge in oxygen biochemistry, is its control. In this work we will investigate how UOX uses O2 to break down uric acid. Interestingly, UOX belongs to a small group of enzymes that can bring oxygen into reacting with their organic substrates and steer the reaction towards the desired products with limited chemical tools at its disposal. In fact, as oxygen activation is not an easy task, the vast majority of enzymes rely on special additional components like metal and/or organic co-factors to form "active oxygen". UOX does not require these additional helpers and therefore understanding how it works is particularly intriguing. Using a technique called X-ray crystallography which allows to 'see' at very high resolution the 3D structure of molecules as small as urate oxidase (10,000 times smaller that the thickness of a human's hair) we have been able to visualise snapshots of the enzyme along the process of uric acid degradation (reaction intermediates) including also the state in which O2 is trapped above the substrate. These snapshots led us to formulate some hypotheses on how urate oxidase works. We are now in an excellent position to study the most elusive and critically important properties of UOX chemistry. For this we will use a technique called neutron crystallography that can detect atoms (hydrogens) that cannot be typically observed even by X-ray crystallography. By combining neutron crystallography, X-ray crystallography, modern spectroscopic techniques and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible we will understand general rules of O2 biochemistry in the context of UOX function. This integrated approach will allow a deeper understanding not only of UOX but also of oxygen, an essential component of life on Earth.