Redox enzymes - tuning and design

项目概要/摘要 酶促氧化还原催化，对所有生物体都是必不可少的，是大自然在调节活性和 在电子水平上的选择性。进行氧化还原转变的金属的还原电位E0为 通过金属的微环境精确地控制在初级配位层之外， 蛋白E0生理相关调节的结果相互作用通常很弱;它们包括 氢键、疏水接触和长程至中程静电。这是一个挑战，研究 这些因素对金属酶氧化还原过程的影响，甚至对设计金属蛋白质的影响 可以进行选择性氧化还原催化。我们提出了一种方法，允许阐述个人 在设计人工钴和锰金属酶的过程中控制金属蛋白氧化还原性质的因素 具有选择性氧还原或氧化反应性。我们提出了金属蛋白构建体，其联合收割机合成 Co和Mn与salen配体的氧化还原活性络合物，以及生物素化的蛋白质链霉亲和素（Sav） 将附着有机金属络合物。在这些系统中，中程静电将被控制 主要在具有独特特征的有机金属络合物内，含有 氧化还原无害金属离子对活性阳离子施加电场。电化学和这些的反应性 将测量和计算复合物。Sav的蛋白质基质将用于整合其他弱 在金属的微环境中的相互作用，如氢键和疏水接触，通过诱变。 混合量子力学和量子经典模拟将指导突变的选择，以考虑 所需的氧化还原活性。重要的是，虽然在天然金属酶中，所有影响E0的弱因素也 影响蛋白质结构并相互耦合，在所提出的系统中，它们在一定程度上是解耦的 这是可能的，因此更适合研究和战略修改。总的来说，这项研究将使学习 氧化还原化学是如何在自然界中控制的，以及如何设计氧化还原酶。我们的目标催化剂 氧还原和需氧底物氧化的反应对天然酶学和生物化学都是有意义的。 更广泛的催化领域，了解这些反应是必不可少的认识如何有氧代谢 有效地利用氧，同时避免形成有害浓度的ROS。

Project Summary/Abstract Enzymatic redox catalysis, imperative to all organisms, is a showcase of nature's mastery in tuning the activity and selectivity at the electronic level. The reduction potential, E0, of the metal performing the redox transformation is precisely controlled beyond the primary coordination sphere through the microenvironment of the metal in the protein. Interactions of consequence for the physiologically relevant modulations of E0 are often weak; they include hydrogen bonds, hydrophobic contacts, and long- to intermediate-range electrostatics. It is a challenge to study the impact of these individual factors on metalloenzymatic redox processes, and even more so to design metalloproteins that would perform selective redox catalysis. We propose an approach that allows elaboration of the individual factors that govern redox properties of metalloproteins en route to the design artificial Co and Mn metalloenzymes with selective oxygen reduction or oxidative reactivity. We propose metalloprotein constructs that combine synthetic redox active complexes of Co and Mn with salen ligands, and the protein streptavidin (Sav) to which biotinylated organometallic complexes will be attached. In these systems, the intermediate-range electrostatics will be controlled largely within the organometallic complex that has a unique feature, a secondary metal binding site containing a redox innocent metal ion exerting an electric field on the active cation. Electrochemistry and reactivity of these complexes will be measured and computed. The protein matrix of Sav will be used for incorporating other weak interactions in the microenvironment of the metal, such as H-bonds and hydrophobic contacts, through mutagenesis. Mixed quantum mechanical and quantum-classical simulations will guide the choice for mutations to consider for the desired redox activity. Importantly, while in natural metalloenzymes all the weak factors influencing E0 also influence the protein structure and couple to each other, in the proposed systems, they are decoupled to the extent possible, and thus more amendable to studying and strategic modifications. Broadly, this research will allow learning how redox chemistry is controlled in nature, and how to approach the design of redox enzymes. Our target catalytic reactions of oxygen reduction and aerobic substrate oxidation are of interest to both natural enzymology and the broader field of catalysis, and understanding these reactions is essential for realizing how aerobic metabolism efficiently utilizes oxygen while avoiding the formation deleterious concentrations of ROS.