Transient and Stable Macromolecular Complexes Formed by Denitrifying Enzymes

氧化还原蛋白，包括金属蛋白，形成蛋白质王国的很大一部分。金属蛋白本身占基因组的30%。这些包含金属离子作为单个原子或作为簇的一部分，并在细菌，植物和动物王国中发挥各种生命维持作用。许多酶利用金属的氧化态来进行氧化还原循环。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。在许多金属酶中，如细胞色素c氧化酶（通过呼吸需求对哺乳动物生命至关重要）、固氮酶和亚硝酸盐还原酶（鉴于其在氮循环中的中心位置而至关重要）、氢化酶（分子氢的生产者-未来替代能源的候选者），催化涉及电子和质子到利用底物的活性位点的受控递送。该提案建立在申请人和RIKEN集团（日本）之间的密切合作的基础上，他们共同在反硝化领域做出了重大贡献，并在我们对涉及金属酶生物机制的复杂过程的理解方面提供了重大进展。我们的综合方法使我们能够在结晶状态下构建详细的三维结构催化电影，从而提供对酶反应化学的详细了解。我们最近确定的膜蛋白的结构，一氧化氮还原酶（Tosha和Shiro，RIKEN）与原子分辨率结构的拴系细胞色素-Cu 1-Cu 2亚硝酸还原酶（Antonyuk，Eady和Hasnain，UoL），使我们在一个非常强大的位置，建立一个综合的结构机械生物学计划。该计划旨在了解全球重要生物系统中氧化还原控制，调节和通信的复杂机制。 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一氧化氮等有毒化学中间体的保护的理解。本计划开发的新方法和途径（例如开发用于研究蛋白质复合物的实验室尺寸排阻色谱-小角X射线散射-动态光散射（SEC-SAXS-DLS））将与我们研究蛋白质复合物的能力具有广泛的相关性。这些新的能力和科学成果将对结构机械生物学产生重大影响，并使英国在这一重要领域处于全球努力的前沿。

Redox proteins, including metalloproteins, form a large portion of the protein kingdom. Metalloproteins themselves form ~ 30% of a genome. These contain metal ions either as a single atom or as part of a cluster and play 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. 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 (essential for mammalian life through respiratory requirements), nitrogenases and nitrite reductases (essential in view of their central position in the nitrogen cycle), hydrogenases (producers of molecular hydrogen - a candidate for a future alternative energy source), catalysis involves the controlled delivery of electrons and protons to the active site where substrate is utilised. The proposal build on close collaboration between the applicants and the RIKEN group (Japan) where they collectively have made major contributions in the field of denitrification and have provided significant advances in our understanding of complex processes that are involved in biological mechanisms of metalloenzymes. Our combined approaches have allowed us to build detailed three dimensional structural movies of catalysis in the crystalline state, thereby providing detailed insight into enzyme reaction chemistry. Our recent determination of structures for the membrane proteins, nitric oxide reducatses (Tosha & Shiro, RIKEN) together with the atomic resolution structure of the tethered cytochrome-Cu1-Cu2 nitrite reductase (Antonyuk, Eady & Hasnain, UoL) puts us in a very strong position to establish an integrated structural-mechanistic biology programme. This programme is aimed at understanding complex mechanisms of redox control, regulation and communication in globally important biological systems. 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 nitric oxide. New methods and approaches developed in this programme (e.g. development of laboratory-based size-exclusion chromatography-small angle X-ray scattering with dynamic light scattering (SEC-SAXS-DLS) for studying protein complexes) will have broad relevance to our capabilities for studying protein complexes. These new capabilities and the scientific outcome will have significant impact on structural-mechanistic biology and keep the UK at the forefront of global effort in this important field.