NextGen Structural Biology under Electrochemical Control: Filling in Missing Intermediates in Metalloenzyme Catalytic Cycles

化学反应对零净可再生能源的未来至关重要，氢气的生产和氧化是一种清洁的可再生燃料，高效生产用于肥料的氨或作为清洁的氢储存系统。大自然已经以微生物氢化酶和氮酶的形式解决了这些化学挑战，它们由地球上丰富的金属簇组成，包裹在蛋白质框架中，使氢作为燃料或从空气中的氮中产生氨成为可能。在这个项目中，我们开发并应用了一套研究工具，这使我们能够填补对这些酶如何工作的理解空白，为更广泛的研究工作提供见解，以建立可行的清洁能源技术，以应对紧迫的气候挑战。我们使用x射线和中子来收集这些酶执行关键催化步骤时的静态图像（类似于“照片”）和动态“电影”的组合，以了解它们如何实现氢和氮中强化学键的分裂。这将提供重要的信息，以帮助生物学家了解酶，并协助化学家设计新的能源技术催化剂。x射线通常用于提供晶体状态下复杂酶分子中原子位置的图像，在晶体状态下，酶的许多分子排列成有序的阵列。酶可以在晶体中进行化学反应，过去十年在同步加速器/激光x射线源和探测器方面取得了令人兴奋的技术进步，这些技术可以快速收集许多x射线“图像”，为制作“电影”提供了可能，这些“电影”显示了酶在起作用时原子是如何运动的。然而，只有当晶体中的所有酶在反应开始时保持在相同的初始状态时，这样的电影才有可能发生——这相当于在比赛前让一群不守规矩的跑步者在起跑线上对齐的挑战——并且所有的酶同时发生反应。这就提出了第二个挑战，找到一个合适的触发器——相当于用来开始比赛的发令枪——来启动反应。我们之前的工作为这些挑战提供了解决方案。首先，我们发现了如何使用电极来施加电化学电位，使所有分子进入一个统一的状态——相同的氧化水平——从而开始催化。其次，Ash已经证明了光触发器可以应用于这种均匀的起始状态来开始催化。在项目中，我们开始微调这些控制和触发机制，使它们适应时间分辨x射线方法中使用的微小晶体。然后，我们使用电化学控制来生成氢化酶每个氧化水平的高质量静态快照。然后，我们应用光触发器来启动催化步骤，并记录酶在作用中的分子电影。这将为氢化酶的实际工作提供迄今为止最详细的视图。接下来，我们解决了x射线结构图像中的一个限制，即很难精确定位酶分解氢气时释放的微小氢原子的位置。为此，我们转向中子束来显示难以捉摸的氢原子。使用非常大的氢化酶晶体，我们再次应用电化学控制将酶分子困在统一的氧化水平，然后向它们发射中子，以显示氢原子的确切位置，这在氢化酶催化中是至关重要的。最后，我们转向氮酶，表明我们也可以将电化学控制和光触发应用于此，证明我们的方法广泛适用于与能源技术相关的不同酶。我们的目标是在非天然底物分子的结合、释放或转化过程中捕捉氮酶的作用，以更好地了解氮在哪里以及如何结合和分裂。

Chemical reactions critical for a net-zero, renewable-energy future are the production and oxidation of hydrogen gas as a clean, renewable fuel, and the efficient production of ammonia for fertiliser or as a clean hydrogen storage system. Nature has already solved these chemical challenges, in the form of microbial hydrogenase and nitrogenase enzymes, which comprise clusters of earth-abundant metals wrapped up in a protein framework to enable use of hydrogen as a fuel or production of ammonia from nitrogen in the air. In this project we develop and apply a set of research tools, which allow us to fill in gaps in understanding of how these enzymes work, providing insight that will feed into wider research efforts to establish viable clean energy technologies to address the urgent climate challenge. We use x-rays and neutrons to collect a combination of static images (akin to 'photographs') and dynamic 'movies' of these enzymes as they carry out key catalytic steps, in order to understand how they achieve the splitting of strong chemical bonds in hydrogen and nitrogen. This will provide important information to assist biologists to understand the enzymes, and to assist chemists to design new catalysts for energy technologies. X-rays are used routinely to provide images of the location of atoms in a complex enzyme molecule in the crystal state, where many molecules of the enzyme pack into an ordered array. Enzymes can perform their chemical reaction in the crystal and the last decade has seen exciting technical advances in synchrotron/laser x-ray sources and detectors that enable rapid collection of many x-ray 'images', offering possibilities of making 'movies' of how atoms move in enzymes as they function. However, such movies are only possible if all the enzymes in the crystal are held in the same initial state at the start of the reaction - equivalent to the challenge of aligning a team of unruly runners at the starting line before a race-and all react at the same time. This presents a second challenge, finding an appropriate trigger- equivalent to a starting gun used to begin a race - to start the reaction. Our previous work provides solutions to these challenges. Firstly, we have found how to use electrodes to apply an electrochemical potential to bring all the molecules into a uniform state - the same oxidation level- to start catalysis. Secondly, Ash has demonstrated light triggers can be applied to this uniform starting state to begin catalysis. During the project, we start by fine-tuning these control and trigger mechanisms, adapting them for the tiny crystals used in time-resolved x-ray methods. We then use electrochemical control to produce high quality static snapshots of each oxidation level of hydrogenase. We then apply the light triggers to initiate steps in catalysis, and record molecular movies of the enzyme in action. This will give the most detailed view ever achieved of hydrogenase actually working.Next, we address a limitation in x-ray structural images that it is very difficult to pinpoint the location of the tiny hydrogen atoms which are released as the enzyme splits hydrogen gas. For this we turn to neutron beams to show up the elusive hydrogen atoms. Using very large crystals of hydrogenase, we again apply electrochemical control to trap the enzyme molecules at a uniform oxidation level, before firing neutrons at them to show the exact positions of the hydrogen atoms that are so critical in hydrogenase catalysis. Finally, we turn to nitrogenase, showing that we can apply our electrochemical control and light triggers here too, demonstrating the broad applicability of our methods to different enzymes relevant to energy technologies. We aim to capture nitrogenase in action during binding, release or transformation of non-natural substrate molecules to better understand where and how nitrogen binds and is split.