Towards a paradigm shift in understanding of membrane-bound Nitric Oxide reductase and its complexes with the electron donor and NO-producing enzyme

膜结合一氧化氮还原酶及其与电子供体和 NO 产生酶复合物的理解的范式转变

• 批准号: BB/X015491/1
• 负责人: Samar Hasnain
• 金额: $ 85.29万
• 依托单位: University of Liverpool
• 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%2FX015491%2F1
• 关键词: Towards; paradigm; shift; understanding; membrane

About one third to one half of all proteins are oxidation/reduction enzymes or metalloproteins. It is estimated that more than one third of all proteins in nature require metals to perform their biological roles and nearly half of all enzymes must associate with a particular metal to function. These metal ions can be either a single atom or form part of a cluster, playing a variety of life-sustaining roles in the bacterial, plant and animal kingdoms. Many enzymes exploit the oxidation states of metals to perform redox cycling during catalysis. Fundamental biological processes in which metalloproteins participate include electron storage and transfer, dioxygen binding, storage and activation, and substrate transport, catalysis and activation. In many metalloenzymes such as cytochrome c oxidase, hydrogenases, nitrogenases and nitrite reductases, catalysis involves the controlled delivery of electrons and protons to the active site where chemical substrates are utilised. These events are often coordinated, coupled and orchestrated by structural signals that remain poorly understood in many cases due to the experimental limitations, particularly membrane proteins, that require solubilization and can be difficult to crystallize. Consequently, although the number of unique structures for membrane proteins has steadily increased since the first structure of a membrane protein in 1985, which brought the Nobel prize in 1988 to Deissenhoffer, Huber and Michel, progress has been slower than predicted. However, recent advances in cryoEM has provided a major boost to structure determination of membrane proteins, catching up the target set in 1990. This project is built on an excellent track record of collaboration and significant underpinning data including the highest resolution structure of any NOR to provide a step change in our understanding of this important membrane metalloenzyme and its complex with NO producing enzyme (AxNiR) that has been studied in our laboratory for several years. The project would provide the first example of protein-protein complexes in catalytic turnover for NOR. This is a challenging project but is highly achievable given our experience and our underpinning data as well as availability of high-quality proteins and several mutants. Our aim is to provide answers to many of the generic questions which are fundamental for (a) protein-protein and protein-ligand interactions, (b) substrate guidance and binding, (c) substrate utilisation with coordinated delivery of electron and proton and (d) product formation and its release. The ability of cryoEM to provide high resolution structure of a frozen solution sample of proteins will enable many of these questions to be addressed, as has been demonstrated very recently for two-component nitrogenases enzyme under turnover conditions (Science 377, 865-869 (2022)). The applicants have an excellent track record of collaboration using cryoEM that has led to several key publications during the last 4 years. Exciting developments arising from our structural and mechanistic work on enzymes catalysing the formation of nitrous oxide by membrane bound quinol-dependent NORs now underpin this timely well-integrated programme where our complementary expertise is harnessed to maintain a world-leading position. General principles emerging from these studies will underpin our understanding of the control of redox processes in biology and protection against toxic chemical intermediates like NO. New methods and approaches that will be developed in this programme will have broad relevance to structural enzymology and keep the UK at the forefront of the global effort. The project would provide a high level of training in membrane structural biology, frontier cryoEM methodology, technology, data processing and structural refinement at high resolution.

所有蛋白质中约三分之一到二分之一是氧化/还原酶或金属蛋白。据估计，自然界中超过三分之一的蛋白质需要金属来发挥其生物学作用，而近一半的酶必须与特定金属结合才能发挥作用。这些金属离子可以是单个原子，也可以是原子簇的一部分，在细菌、植物和动物界中发挥着多种维持生命的作用。许多酶利用金属的氧化态在催化过程中进行氧化还原循环。金属蛋白参与的基本生物过程包括电子存储和转移、分子氧结合、存储和活化以及底物传输、催化和活化。在许多金属酶（例如细胞色素 C 氧化酶、氢化酶、固氮酶和亚硝酸还原酶）中，催化作用涉及将电子和质子受控传递到利用化学底物的活性位点。这些事件通常是由结构信号协调、耦合和编排的，由于实验的限制，在许多情况下人们对结构信号的了解仍然知之甚少，特别是膜蛋白，需要溶解并且很难结晶。因此，尽管自 1985 年首次提出膜蛋白结构以来，膜蛋白独特结构的数量一直在稳步增加，并为戴森霍夫、胡贝尔和米歇尔带来了 1988 年诺贝尔奖，但进展速度却比预期要慢。然而，冷冻电镜的最新进展极大地促进了膜蛋白的结构测定，赶上了 1990 年设定的目标。该项目建立在良好的合作记录和重要的基础数据的基础上，包括任何 NOR 的最高分辨率结构使我们对这种重要的膜金属酶及其与 NO 产生酶 (AxNiR) 的复合物的理解发生了重大变化，我们的实验室已对这种酶进行了多年的研究。该项目将提供蛋白质-蛋白质复合物在 NOR 催化转化中的第一个例子。这是一个具有挑战性的项目，但鉴于我们的经验和基础数据以及高质量蛋白质和几种突变体的可用性，这是非常可以实现的。我们的目标是为许多通用问题提供答案，这些问题对于以下方面至关重要：（a）蛋白质-蛋白质和蛋白质-配体相互作用，（b）底物引导和结合，（c）协调电子和质子传递的底物利用以及（ d) 产品的形成及其释放。冷冻电镜能够提供蛋白质冷冻溶液样品的高分辨率结构，这将使许多这些问题得到解决，正如最近在周转条件下的双组分固氮酶所证明的那样（Science 377, 865-869 (2022 ））。申请人在使用冷冻电镜方面拥有良好的合作记录，在过去 4 年中发表了多篇重要出版物。我们在通过膜结合的对苯二酚依赖性 NOR 催化一氧化二氮形成的酶方面的结构和机械工作取得了令人兴奋的进展，现在支撑了这一及时的良好整合计划，在该计划中，我们利用互补的专业知识来保持世界领先地位。这些研究得出的一般原理将巩固我们对生物学中氧化还原过程的控制以及对有毒化学中间体（如一氧化氮）的防护的理解。该计划将开发的新方法和途径将与结构酶学具有广泛的相关性，并使英国保持在全球努力的前沿。该项目将提供膜结构生物学、前沿冷冻电镜方法、技术、数据处理和高分辨率结构细化方面的高水平培训。