Investigation of Water Oxidizing Catalysis for Renewable Energy

化石燃料的燃烧释放出二氧化碳，几乎可以肯定这是人为气候变化的原因。因此，我们必须找到替代的“碳中和”能源作为当务之急。到目前为止，最大的潜在可再生能源是阳光。利用这种能源是我们文明面临的巨大挑战之一，但使用它是有问题的。现有的硅太阳能电池价格昂贵，效率低下，而且不能产生燃料。植物已经找到了完美的解决方案；它们利用阳光的能量来氧化水(H2O)，释放出O2、质子和电子。电子和质子被用来将二氧化碳固定为有机糖，然后这些有机糖可以用于生物合成或用作呼吸作用的燃料。整个过程被称为光合作用。植物可以被种植来生产所谓的生物燃料，如生物柴油，但这是低效的，并与粮食生产形成竞争。需要的是一种人工光合作用系统，它像植物一样，将阳光、水和二氧化碳转化为燃料，但成本低、效率高，可以大面积部署。拟议的项目是创造一个未来太阳能转换系统的重要组成部分。光合作用装置有许多组成部分，但这个项目最感兴趣的是一种名为光系统II(PSII)的酶。PSII负责光合作用的光驱动水分解反应。在其核心，PSII有一个由一个钙离子和四个锰离子组成的簇，这些离子催化了水的分解反应。这个星团被称为放氧中心(OEC)。OEC的确切结构和作用机制尚不清楚，但如果要构建合成的光驱动水氧化酶，必须了解这两个方面。建立这样的系统是高效大规模利用太阳能的重要前提。PSII中的OEC很难研究，因为PSII是一个包含许多蛋白质分子和辅因子以及OEC的大型复合体。因此，我建议使用小蛋白作为锰离子的支架，从而构建一种与PSII酶解偶联的OEC类似物，它可以更容易地进行研究，是未来设备的现实原型。除了PSII，还有许多已知的酶在其活性部位含有两个锰离子，但PSII的独特之处在于一个位点有四个锰离子。我想用这些更简单的锰酶中的一种来改造它来结合更多的锰离子，模仿PSII。这可以通过重组DNA技术来实现。构建了含有编码所设计的酶的序列的DNA分子，然后将其导入无害细菌中。然后诱导细菌产生修饰的酶，然后提取和纯化以供进一步研究。这项技术的优点是DNA分子易于操纵，可以快速且廉价地产生特定序列，允许在短时间内尝试多种酶设计。在产生了与多个锰离子结合的修饰蛋白质分子后，可以确定三维结构。将对该蛋白质进行类似于PSII的酶活性的探测。我将尝试使用强氧化剂作为光的替代品来催化水或其他底物的氧化。在植物中，叶绿素是主要的光敏色素，但在人工系统中，叶绿素通常是不稳定的。相反，我会将稳定的合成色素偶联到蛋白质上，并尝试利用光来产生氧化反应。这些实验的结果可以与酶的三维结构相关，然后被用来为工程蛋白质的设计提供修改的信息，然后将接受进一步的实验和设计。这种“人工选择进化”可以反复进行，直到实现了可溶性水氧化酶的预期目标。

The burning of fossil fuels releases CO2 which is almost certainly responsible for anthropogenic climate change. Therefore, we must find alternative 'carbon-neutral' sources of energy as a matter of urgency. By far the largest potential source of renewable energy is sunlight. Harnessing this energy is one of the great challenges that our civilization faces, but using it is problematic. Existing silicon solar cells are expensive and inefficient, and do not produce fuel. Plants have hit on the perfect solution; they use the energy of sunlight to oxidise water (H2O), liberating O2, protons and electrons. The electrons and protons are used to fix carbon dioxide as organic sugars, which may then be used for biosynthesis or as fuel for respiration. The total process is known as photosynthesis. Plants can be grown to generate so-called 'biofuels' such as biodiesel, but this is inefficient, and competes with food production. What is needed is an artificial photosynthetic system, that, like plants, converts sunlight, water and CO2 into fuel, but is cheap, efficient and can be deployed over large areas. The proposed project is to create a vital component of a future solar energy conversion system. There are many components to the photosynthetic apparatus, but the main one of interest to this project is an enzyme called photosystem II (PSII). PSII is responsible for the light-driven water splitting reaction of photosynthesis. At its core, PSII has a cluster of one calcium and four manganese ions, which catalyse the water splitting reaction. This cluster is known as the oxygen evolving centre (OEC). The precise structure of the OEC and the mechanism of its action are still unknown, but both of these must be understood if a synthetic light-driven water oxidase is to be constructed. Building such a system is a vital prerequisite for the efficient large scale use of solar energy. The OEC in PSII is difficult to study, as PSII is a large complex containing many protein molecules and cofactors as well as the OEC. Therefore I propose to use small proteins as scaffolds for manganese ions, and so construct an OEC analogue that is uncoupled from the PSII enzyme and can be studied much more easily, and is a realistic prototype for future devices. Apart from PSII, there are many known enzymes which contain two manganese ions at their active sites, but PSII is unique in having four manganese ions at one site. I would like to take one of these simpler manganese enzymes and engineer it to bind more manganese ions, to mimic PSII. This can be accomplished by recombinant DNA technology. A DNA molecule with a sequence encoding the designed enzyme is constructed and then introduced into a harmless bacterium. The bacterium is then induced to produce the modified enzyme, which is then extracted and purified for further study. This technique has the advantage that DNA molecules are easy to manipulate, and specific sequences can be produced quickly and cheaply, allowing many designs of enzyme to be tried in a short time. Having produced a modified protein molecule that binds multiple manganese ions, the three dimensional structure can be determined. The protein will be probed for enzyme activity similar to that of PSII. I will try to catalyse the oxidation of water or other substrates using powerful oxidants as a substitute for light. In plants, chlorophyll is used as the main photosensitive pigment, but chlorophyll is usually unstable in artificial systems. Instead I will couple stable synthetic pigments to the protein and try to generate oxidative reactions using light. The results of these experiments can be related to the three dimensional structure of the enzyme and then used to to inform modifications in the design of the engineered proteins, which will then be subjected to further rounds of experimentation and design. This 'evolution by artificial selection', can be iterated until the desired goal of a soluble water oxidase is realised.