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.

大约三分之一到一半的蛋白质是氧化/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，几乎一半的酶必须与特定的金属结合才能发挥作用。这些金属离子可以是单个原子，也可以是簇的一部分，在细菌、植物和动物王国中扮演着各种维持生命的角色。许多酶在催化过程中利用金属的氧化状态来执行氧化还原循环。金属蛋白参与的基本生物学过程包括电子储存和传递、氧结合、储存和活化、底物运输、催化和活化。在许多金属酶中，如细胞色素C氧化酶、氢酶、固氮酶和亚硝酸盐还原酶，催化涉及到电子和质子被控制地传递到利用化学底物的活性部位。这些事件通常是由结构信号协调、耦合和协调的，而在许多情况下，由于实验的限制，特别是膜蛋白，这些信号仍然知之甚少，需要增溶，并且可能难以结晶。因此，尽管膜蛋白的独特结构的数量自1985年膜蛋白的第一个结构以来一直在稳步增加，这使得1988年的诺贝尔奖授予了Deissenhoffer，Huber和Michel，但进展比预期的要慢。然而，低温电子显微镜的最新进展为膜蛋白的结构测定提供了重大的推动，赶上了1990年设定的目标。这个项目建立在出色的合作记录和重要的基础数据基础上，包括最高的分辨率结构，也不是为了让我们对这种重要的膜金属酶及其与无产酶的络合物(AxNiR)的理解发生阶段性变化，我们实验室已经研究了几年。该项目将为NOR提供第一个蛋白质-蛋白质复合体催化周转的例子。这是一个具有挑战性的项目，但考虑到我们的经验和基础数据，以及高质量蛋白质和几个突变体的可用性，这是非常有可能实现的。我们的目标是为许多一般性问题提供答案，这些问题对于(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)底物的利用与电子和质子的协调传递以及(D)产物的形成和释放是基本的。低温EM提供蛋白质冷冻溶液样本的高分辨率结构的能力将使这些问题得以解决，就像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))。申请者在使用CryoEM方面有着良好的合作记录，在过去的4年里已经发表了几篇重要的论文。我们在酶的结构和机制方面的工作取得了令人兴奋的发展，这些酶通过膜结合的依赖于苯酚的NORs催化形成一氧化二氮，现在支撑着这一及时而良好的综合计划，其中我们的互补专业知识被利用来保持世界领先地位。从这些研究中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和对NO等有毒化学中间体的保护的理解。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。该项目将在膜结构生物学、前沿低温电磁方法、技术、数据处理和高分辨率结构改进方面提供高水平的培训。