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

电化学控制下的下一代结构生物学：填补金属酶催化循环中缺失的中间体

基本信息

批准号：
BB/X002624/1
负责人：
Kylie Vincent
金额：
$ 73.59万
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FX002624%2F1
关键词：
NextGen Structural Biology under Electrochemical

项目摘要

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.

对于零零，可再生能源的未来至关重要的化学反应是氢气作为干净，可再生燃料的生产和氧化，以及用于肥料或清洁氢存储系统的有效产生。大自然已经以微生物氢化酶和硝化酶的形式解决了这些化学挑战，这些酶包括包裹在蛋白质框架中的地球量的金属簇，以使氢用作空气中氮气的燃料或氨的产生。在这个项目中，我们开发并应用了一套研究工具，使我们能够填补这些酶如何工作的空白，提供洞察力，这些洞察力将介入更广泛的研究工作，以建立可行的清洁能源技术，以应对紧急气候挑战。我们使用X射线和中子来收集这些酶执行关键催化步骤的这些酶的静态图像（类似于“照片”）和动态的“电影”的组合，以了解它们如何实现氢和氮气中强化学键的分裂。这将提供重要的信息，以帮助生物学家了解酶，并协助化学家为能量技术设计新的催化剂。 X射线通常用于提供在晶体状态的复杂酶分子中原子位置的图像，其中许多酶包的分子成有序的阵列。酶可以在晶体中进行化学反应，而在过去的十年中，酶在同步加速器/激光X射线源和检测器中的技术进步令人兴奋，可以快速收集许多X射线“图像”，从而提供了制作原子如何在酶中移动原子的可能性的可能性。但是，只有在反应开始时，晶体中的所有酶都以相同的初始状态保持，这是可能的，这相当于挑战在比赛之前将不守规矩的跑步者在起跑线上与一支不守规矩的跑步者保持一致，并且所有人都在同时做出反应。这提出了第二个挑战，找到了一个适当的触发因素，等同于开始比赛的起始枪 - 开始反应。我们以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应用电化势将所有分子带入均匀状态 - 相同的氧化水平 - 开始催化。其次，灰烬已证明可以将光触发器应用于这种均匀的起始状态以开始催化。在项目期间，我们从微调这些控制和触发机制开始，将它们调整为在时间分辨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产生每种氧化氢酶氧化水平的高质量静态快照。然后，我们将光触发器应用于催化中的步骤，并记录酶的分子电影。这将使有史以来最详细的氢化酶实际上可以实现的视图。接下来，我们解决了X射线结构图像的限制，即很难确定小氢原子的位置，这些氢原子被释放，这些氢原子被释放而释放。为此，我们转向中子束以显示难以捉摸的氢原子。使用非常大的氢化氢晶体，我们再次应用电化学对照，以均匀的氧化水平将酶分子捕获，然后向其发射中子以显示氢原子的确切位置，这些位置在氢酶催化中至关重要。最后，我们转向硝基化酶，表明我们也可以在这里应用电化学控制和光触发器，证明我们方法在与能量技术相关的不同酶中的广泛适用性。我们旨在捕获非天然底物分子的结合，释放或转化过程中的作用中的氮酶，以更好地了解氮的结合和分裂方式。