Acceleration and control of spin-restricted oxygenation by cofactor-independent dioxygenases

人类和所有其他高度进化的生物一样，严格依赖大气中的氧气来生存。通过呼吸过程获得的氧气对于产生我们生理功能所需的能量以及防御各种感染是必不可少的。氧气也用于各种有机化合物的降解，一些细菌利用它来帮助分解环境污染物分子。这要归功于一种叫做加氧酶的特殊酶的作用，这种酶能够促进氧原子与分子结合的反应，否则分子就很难被处理掉。加氧酶的任务很困难，因为氧在正常的“静息”状态下（空气中的形式），由于其电子结构的原因，不愿与绝大多数分子发生反应。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）是有害的“活性氧”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。激活和控制是氧依赖性生物过程的关键。在这项工作中，我们将研究两种细菌加氧酶，它们分别被称为HOD和QDO，它们与其他加氧酶组成一个独立的家族。有趣的是，它们可以将氧气带入与有机底物的反应中（激活），并使用有限的工具将反应导向所需的产物（控制）。事实上，由于氧气活化不是一件容易的事，绝大多数加氧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”。HOD和QDO不具备这些额外的功能，因此了解它们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，它使我们能够以非常高的分辨率可视化像HOD和QDO这样小的分子的3D结构（它们比人类头发的厚度小大约一万倍），我们现在详细地知道了这些酶的形状。它们看起来不像其他已知的双加氧酶；相反，它们具有另一种酶家族的结构，这种酶家族通常催化不涉及氧气的反应。使用相同的x射线技术，我们也看到了底物在没有氧气的情况下与HOD结合的位置，以及它与酶的具体相互作用。同样，我们已经看到了反应产物在离开酶进入新的反应循环之前是如何结合的。这些快照使我们对HOD/QDO如何工作提出了一些假设。我们现在处于一个很好的位置，可以研究HOD/QDO如何工作的最有趣的方面。一方面是氧气被激活和控制将底物转化为产物的步骤另一方面是允许通常用于不同反应的蛋白质支架在这里被用来承载氧气生物化学的原因。我们将再次使用x射线晶体学来可视化氧与这些酶的结合，使用现代光谱技术来研究反应周期不同阶段的重要电子性质，并使用先进的量子力学理论方法来探测实验无法获得的状态。这种多角度的方法将使我们对氧气的生物学有新的认识，氧气是地球上生命的重要组成部分。

Humans like all other highly evolved organisms strictly depend on atmospheric oxygen for survival. Oxygen obtained via the respiration process is essential for the production of energy required to carry out our physiological functions as well as for the defence against various kinds of infections. Oxygen is also used for the degradation of various organic compounds and some bacteria use it to help breakdown molecules that are environmental pollutants. This is thanks to the action of particular enzymes, called oxygenases, which are able to promote reactions in which oxygen atoms are incorporated into molecules otherwise difficult to dispose of. The task of oxygenases is a difficult one because oxygen in its normal 'resting' state (the form present in the air) does not want to react with the vast majority of 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. ACTIVATION and CONTROL are critical keywords in oxygen-dependent biological processes. In this work we will investigate two bacterial oxygenases called with the acronyms of HOD and QDO which constitute a separate family from other oxygenases. Interestingly, they can bring oxygen into reactions with their organic substrates (ACTIVATION) and steer the reaction towards the desired products (CONTROL) with limited tools at their disposals. In fact, as oxygen activation is not an easy task, the vast majority of oxygenases rely on special additional components like metal and/or organic co-factors to form 'active oxygen'. HOD and QDO don't possess these additional features and therefore understanding how they work is particularly intriguing. Using a technique called X-ray crystallography which allows us to visualise at very high resolution the 3D structure of molecules as small as HOD and QDO (they are about ten thousand times smaller that the thickness of a human's hair) we now know in detail the shape of these enzymes. They do not look like other known dioxygenases; rather they have an architecture of another enzyme family which typically catalyses reactions not involving oxygen. Using the same X-ray technique we have also seen where the substrate binds to HOD when oxygen is not around and what specific interactions it makes with the enzyme. Similarly, we have seen how the reaction product is bound before leaving the enzyme for a new reaction cycle. These snapshots led us to formulate some hypotheses on how HOD/QDO work. We are now in an excellent position to study the most interesting aspects of how HOD/QDO work. These are on one hand the steps in which oxygen gets ACTIVATED and CONTROLLED to convert the substrate into products and on the other hand the reasons which allow a protein scaffold used typically for different reactions to be used here to host oxygen biochemistry. We will again use X-ray crystallography to visualise oxygen bound to the these enzymes, modern spectroscopic techniques to study important electronic properties at different stages of the reaction cycle, and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible. This multi-angle approach will allow novel insights into the biology of oxygen, an essential component of life on Earth.