Transient and Stable Macromolecular Complexes Formed by Denitrifying Enzymes

氧化还原蛋白，包括金属蛋白，构成了蛋白质王国的很大一部分。金属蛋白本身约占基因组的30%。它们含有金属离子，要么作为单个原子，要么作为簇的一部分，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、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(通过呼吸需求对哺乳动物的生命至关重要)、固氮酶和亚硝酸盐还原酶(鉴于它们在氮循环中的中心地位而必不可少)、氢酶(产生分子氢--未来替代能源的候选者)，催化涉及到电子和质子被控制地输送到底物被利用的活性部位。该提案建立在申请者和理研集团(日本)之间的密切合作基础上，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取得了重大进展。我们的组合方法使我们能够构建结晶状态下催化的详细三维结构电影，从而提供对酶反应化学的详细见解。我们最近对膜蛋白、一氧化氮还原酶(Tosha&Shiro，RIKEN)的结构的测定，以及束缚的细胞色素-Cu1-Cu2亚硝酸盐还原酶(Antonyuk，Eady&Hasnain，UOL)的原子分辨结构，使我们处于建立综合结构-机械生物学计划的非常有利的地位。该方案旨在了解全球重要生物系统中氧化还原控制、调节和交流的复杂机制。从这些研究中得出的一般原则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)的保护的理解。这项计划中开发的新方法和途径(例如，用于研究蛋白质复合体的基于实验室的尺寸排除层析-小角X射线散射和动态光散射(SEC-SAXS-DLS))将与我们研究蛋白质复合体的能力广泛相关。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，并使英国保持在这一重要领域的全球努力的前沿。

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.