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

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，近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学过程包括电子储存和传递、分子氧结合、储存和活化以及底物转运、催化和活化。在许多金属酶如细胞色素c氧化酶、氢化酶、固氮酶和亚硝酸盐还原酶中，催化涉及电子和质子到利用化学底物的活性位点的受控递送。这些事件往往是协调，耦合和精心安排的结构信号，在许多情况下，由于实验的限制，仍然知之甚少，特别是膜蛋白，需要溶解，并可能难以结晶。因此，尽管自1985年发现第一个膜蛋白结构以来，膜蛋白独特结构的数量稳步增加，并于1988年将诺贝尔奖授予Deissenhoffer，Huber和Michel，但进展比预期的要慢。然而，cryoEM的最新进展为膜蛋白的结构测定提供了重大推动，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之上，包括任何NOR的最高分辨率结构，以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变化，该酶已在我们的实验室研究了数年。该项目将提供第一个蛋白质-蛋白质复合物催化NOR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种突变体的可用性，这是高度可实现的。我们的目的是提供答案的许多通用的问题，这是基本的（a）蛋白质-蛋白质和蛋白质-配体相互作用，（B）基板的指导和结合，（c）基板利用与协调传递的电子和质子和（d）产品的形成和释放。cryoEM提供蛋白质冷冻溶液样品的高分辨率结构的能力将使这些问题中的许多问题得以解决，正如最近在周转条件下对双组分固氮酶所证明的那样（Science 377，865-869（2022））。申请人在使用cryoEM的合作方面有着良好的记录，在过去4年中已经发表了几篇重要论文。我们对酶催化膜结合的喹啉依赖性NORs形成一氧化二氮的结构和机制工作所产生的令人兴奋的发展现在支持了这个及时的综合计划，利用我们的互补专业知识来保持世界领先地位。从这些研究中出现的一般原则将巩固我们对生物学中氧化还原过程的控制和对有毒化学中间体（如NO）的保护的理解。将在该计划中开发的新方法和方法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最前沿。该项目将在膜结构生物学、前沿cryoEM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。

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.