Investigation of Water Oxidizing Catalysis for Renewable Energy

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.

Solar energy is the only feasible source of renewable energy that can replace fossil fuels. To develop sunlight as a viable source of energy it will be necessary to imitate the light reactions of photosynthesis. Photosystem II (PSII) is the only known system capable of using visible light to oxidise water to oxygen, releasing reducing equivalents, which can then be used to fix CO2 and generate fuel. The reaction is catalysed at a cluster of manganese ions and other associated cofactors. Despite crystal structures of PSII at up to 3.0A and characterization of the complex by other biophysical techniques, the structure of the cluster is still under debate. I propose to create a multiple manganese cluster, in imitation of PSII, using existing and de novo proteins as scaffolds into which the cluster is assembled. Such a cluster will be much easier to study than the OEC of PSII, which is part of a monolithic membrane protein complex. The synthetic clusters will be characterised by X-ray crystallography and EXAFS to confirm the location and incorporation of manganese ions. The protein design will be driven by knowledge of the rotameric properties of amino acids and proteins and statistical information from the protein structure and small molecule databases on the binding modes of manganese ions. A strong candidate protein for the initial design is the C. trachomatis ribonucleotide reductase. This protein has a Mn(IV)-Fe(III) site, which has the only known biological high valency manganese outside PSII. Therefore it is an ideal substrate into which more Mn sites can be introduced, having an existing Mn(IV) and a four-helix bundle architecture into which amino acid changes can be made. The engineered proteins will be expressed in E. coli. After a manganese cluster has been produced, I will attempt to generate oxidative chemistry using both chemical oxidants such as potassium peroxymonosulfate, and reactions driven by pigments, such as ruthenium polypyridines.