Collaborative Research: Design of Redox-Active Molybdenum Metalloproteins

项目编号：1403532 / 1403663PI’s: Zondlo, Neal J. / Kuhlman， brian机构：特拉华大学/北卡罗来纳大学教堂山分校标题：合作研究：氧化还原活性钼金属蛋白的设计氮是DNA和蛋白质的重要组成部分，是所有生命形式所必需的。然而，大多数生物不能利用占大气78%的氮气（N2）。在被植物和动物代谢之前，氮气必须被“固定”为氨（NH3）等还原形式。这个过程在自然界中是由与植物共生的细菌完成的。通过Haber-Bosch工艺在高温高压下进行的合成固氮是化肥生产的核心，使地球能够维持比以前更多的人口，而代价是地球总能源消耗的1.5%。固氮酶是一种含钼的蛋白质，被固氮细菌利用，在室温和常压下在水中完成固氮，这与哈伯-博世工艺的苛刻和能源密集型条件形成了对比。氮酶还能够与二氧化碳（即进行碳固存）和一氧化碳反应，将它们还原成可用作液体燃料和原料化学品的分子。然而，氮酶是大蛋白质（大约2000个氨基酸），这阻碍了它们作为分离蛋白质的应用。在这项工作中，研究人员将开发合成蛋白质，其大小仅为氮酶的2%-4%，可以结合钼并以类似于氮酶的方式与分子反应。这项工作的基础是开发新型氨基酸，既可以与钼结合，又可以向金属提供电子，以参与钼金属中心的反应化学，结合计算蛋白质设计，允许将这些非天然氨基酸结合在一个定义良好且稳定的蛋白质结构中，这将支持强金属结合和反应化学。这些设计的钼蛋白将被分析和表征其结构和进行反应化学的能力，以了解天然钼蛋白如何进行重要的反应，并作为其在能源使用问题关键过程中潜在应用的第一步。这项工作将解决氮循环、碳循环、能源和可持续性的关键过程，为广泛的基础重要性过程提供新的见解。从长远来看，这项工作可能会带来新的可持续解决方案，以减少能源使用，并从非石油来源合成原料化学品。这项工作将培养本科生和研究生在高度多学科的方法，包括计算蛋白设计，多肽合成和表征，富功能基底物上的有机合成，培养他们为21世纪的综合多学科科学。固氮作用是将大气中的氮还原为氨，是地球上最重要的过程之一。固氮是由含有固氮酶的细菌完成的，固氮酶是一种含有钼或钒和独特的铁硫簇的大型（约2000个氨基酸）酶。在温和的条件下完成这个古老的过程，迄今为止还没有在天然的氮酶蛋白之外的蛋白质中实现。作为启发，用小分子配体合成钼配合物证明了催化固氮。为了开发具有氧化还原活性的蛋白质，使其具有类似于氮酶的反应化学功能，该团队将开发一种合成的钼金属蛋白，该蛋白含有新型的氧化还原活性氨基酸，比天然的20种氨基酸具有更大的电子供体能力。氮酶和合成类似物完成二氮还原（六电子，六质子过程），部分原因是钼(Mo（III）到Mo（VI）的多种氧化态，加上强电子供体配体的存在，包括铁硫碳化物簇。氮酶和有机钼配合物也能还原电子相关的化合物，如二亚胺（包括肼HN=NH）、肼（H2N-NH2）和氰化物。这项工作旨在开发新的方法，允许使用合成蛋白质减少简单的化合物，并为催化活性蛋白质的设计提供基本的见解。研究人员将设计具有氧化还原活性侧链的合成蛋白质，这种蛋白质在天然存在的蛋白质中没有发现，并且很容易与钼、钒和钨结合，从而允许多电子还原二氮和相关分子。这些设计的蛋白质的金属结合特性将使用电化学、生物物理和结构方法在不同的金属氧化还原状态下进行表征。所设计的具有氧化还原活性的金属蛋白将被检测对一系列pi键合化合物的还原反应性。该奖项由CBET部门的生物技术、生化和生物质工程项目颁发，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

Proposal Numbers: 1403532 / 1403663PI's: Zondlo, Neal J. / Kuhlman, BrianInstitutions: University of Delaware / University of North Carolina at Chapel HillTitle: Collaborative Research: Design of Redox-Active Molybdenum MetalloproteinsNitrogen is a critical component of DNA and proteins and is essential for all forms of life. However, most organisms cannot make use of the nitrogen gas (N2) that makes up 78% of our atmosphere. Nitrogen gas must be "fixed" to reduced forms such as ammonia (NH3) before it can be metabolized by plants and animals. This process is accomplished in nature by bacteria that live symbiotically with plants. Synthetic nitrogen fixation, performed at high temperatures and high pressures via the Haber-Bosch process, is central to the production of fertilizers, allowing the planet to sustain far larger populations than was possible prior, at the cost of 1.5% of the planet's total energy use. Nitrogenases are molybdenum-containing proteins employed by nitrogen-fixing bacteria to accomplish nitrogen fixation in water at ambient temperature and pressure, in contrast to the harsh and energy-intensive conditions of the Haber-Bosch process. Nitrogenases are also capable of reacting with carbon dioxide (i.e. conducting carbon sequestration) and carbon monoxide, reducing them to molecules that can be used as liquid fuels and feedstock chemicals. However, nitrogenases are large proteins (approximately 2000 amino acids), which prevents their application as isolated proteins. In this work, the investigators will develop synthetic proteins, only 2%-4% of the size of nitrogenase, that can bind molybdenum and react with molecules in a manner analogous to that of nitrogenases. The basis of this work is the development of novel amino acids that can both bind to molybdenum and donate electrons to the metal to engage in reaction chemistry at the molybdenum metal center, combined with computational protein design to allow the incorporation of these unnatural amino acids within a well-defined and stable protein structure that will support strong metal binding and reaction chemistry. These designed molybdenum proteins will be analyzed and characterized for their structure and ability to conduct reaction chemistry, both to understand how native molybdenum proteins can conduct important reactions and as a first step toward their potential use in processes critical to problems in energy use. This work will address critical processes central to the nitrogen cycle, the carbon cycle, energy, and sustainability, providing new insights into processes of broad fundamental importance. In the long term, this work could lead toward novel sustainable solutions to reduce energy use and synthesize feedstock chemicals from non-petroleum sources. This work will train undergraduate and graduate students in highly multidisciplinary methods, including computational protein design, peptide synthesis and characterization, organic synthesis on functional-group rich substrates, training them for integrated multidisciplinary science of the 21st century. Nitrogen fixation, the reduction of atmospheric nitrogen to ammonia, is one of the most significant processes on the planet. Nitrogen fixation is accomplished by bacteria containing the enzyme nitrogenase, a large (~2000 amino acids) enzyme containing molybdenum or vanadium and a unique iron-sulfur cluster. The accomplishment of this ancient process under mild conditions has to date never been achieved in proteins outside the native nitrogenase proteins. As inspiration, catalytic nitrogen fixation has been demonstrated using synthetic molybdenum complexes with small molecule ligands. Toward the goal of developing redox-active proteins capable of reaction chemistry similar to nitrogenase, the team will develop a synthetic molybdenum metalloprotein containing novel redox-active amino acids capable of greater electron donor ability than the native 20 amino acids. Nitrogenases and synthetic analogues accomplish dinitrogen reduction (a six-electron, six-proton process) in part due to the multiple oxidation states readily available to molybdenum (Mo(III) to Mo(VI)), plus the presence of strong electron donor ligands, including the iron-sulfur-carbide cluster. Nitrogenases and organomolybdenum complexes can also reduce electronically related compounds such as diimides (including diazine HN=NH), hydrazines (H2N-NH2), and cyanide. This work aims to develop new approaches to allow the reduction of simple compounds using synthetic proteins and provide fundamental insights into the design of catalytically active proteins. The investigators will design synthetic proteins with redox-active side chains that are not found in naturally occurring proteins and readily bind to molybdenum, vanadium and tungsten, allowing multi-electron reductions of dinitrogen and related molecules. The metal-binding properties of these designed proteins will be characterized in different metal redox states using electrochemical, biophysical, and structural methods. The designed redox-active metalloproteins will be examined for reactivity toward reduction of a series of pi-bonded compounds.This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.