De novo designed metalloproteins as a new generation of artificial hydrogenases

项目摘要/摘要 氢酶是一种复杂的、含有金属的酶，通过以下方式为某些生物体产生能量 催化H+和H2气体之间的可逆相互转化。揭开关于这一事件的复杂细节 这些酶的功能将显著推动氢基、碳中性替代能源的发展 制作。然而，这些酶的复杂性由于存在多个金属辅因子，低 产量和失活，使研究这些酶具有挑战性。我们的长期目标是 设计人工生物分子氢酶(ARHS)作为这些金属酶的更简单的功能类似物。 从头开始的金属蛋白设计是一种很有吸引力的和成熟的模型复合体方法 最小蛋白支架中的金属蛋白。虽然设计的系统不那么复杂，但它们服务于 作为天然金属酶的水溶性功能类似物，并提供了 化学反应。采用这种方法，我们建议追求描述总体设计的三个具体目标 受[NiFe]氢酶启发的ARH的原理和功能/机械属性。整体而言 该方案的目标是：i)设计活性的单核(Ni)、双核(Ni-Fe)和多核(Ni3) 合适的从头开始的支架中的位置；ii)表征arhs的物理和催化性能；iii) 确定电子转移的时间尺度；iv)概述H+转移途径；v)表征反应 以及vi)阐明金属和蛋白质支架如何协同工作以影响 性质/反应性，从而可以获得H-H键形成的整体机械观点。我们的强者 这里介绍的初步结果证明，我们的目标是可以实现的。 总的来说，这项拟议工作的结果将影响金属蛋白设计、生物无机等领域 化学和替代能源研究。将出现一类新的ARH，它将提供功能 重点介绍了与天然酶有关的H+还原的工作原理。模块化设计 这项研究的参数和结果将使我们能够制备具有新性能的生物合成催化剂 并在未来发挥作用。

PROJECT SUMMARY/ABSTRACT Hydrogenases are complex, metal-containing enzymes that generate energy for certain organisms by catalyzing the reversible interconversion between H+ and H2 gas. Unraveling the intricate details about the function of these enzymes will significantly advance the H2-based, carbon-neutral alternative energy production. However, the complexity of these enzymes due to the presence of multiple metallic cofactors, low production yield, and deactivation, makes studying these enzymes challenging. Our long term goals are to design artificial biomolecular hydrogenases (ArHs) as simpler functional analogs of these metalloenzymes. De novo metalloprotein design is an appealing and well-established approach to model complex metalloproteins within minimal protein scaffolds. Although the designed systems are less complex, they serve as water-soluble functional analogs of the native metalloenzymes and provide a functional view of the chemistry. Employing this approach, we propose to pursue three Specific Aims describing the overall design principles and functional/mechanistic attributes of the ArHs inspired by the [NiFe] hydrogenases. The overall objectives of this proposal are: i) to design mononuclear (Ni), binuclear (Ni-Fe), and multinuclear (Ni3) active sites within suitable de novo scaffolds; ii) characterize the physical and catalytic properties of the ArHs; iii) determine the timescales of electron transfer; iv) outline the H+ transfer pathways; v) characterize the reaction intermediates; and vi) elucidate how metals and protein scaffold work in synergy to influence the properties/reactivity, such that a holistic mechanistic view of H-H bond formation can be attained. Our strong preliminary results presented here attest that our objectives are achievable. Collectively, the results from this proposed work will impact the fields of metalloprotein design, bioinorganic chemistry, and alternative energy research. A novel class of ArHs will emerge, which will provide functional vignettes into the working principles of H+ reduction pertaining to the native enzymes. The modular design parameters and outcomes from this study will enable us to prepare biosynthetic catalysts with novel properties and functions in the future.