Investigating the early steps in the assembly of the oxygen-evolving complex of photosynthesis

The photosystem two (PSII) protein complex is widely considered to be one of the most remarkable and important molecular machines on Earth. It performs the incredibly difficult task of extracting electrons from highly stable water molecules to allow plants, algae and cyanobacteria to grow. PSII also produces the oxygen that we breathe. PSII drives the very demanding water-splitting reaction by capturing solar energy and using it to drive the oxidation of water molecules bound to a highly conserved metal cluster, made up of 1 calcium ion and 4 manganese ions, buried within the PSII complex. The oxygen that is liberated is then fed back into the atmosphere. Dramatic progress has been made in understanding the 3-dimensional structure of the complex so that we now know to a high degree of precision where each atom in the complex is located. We know that active PSII is composed of about 20 individual proteins, bound together in a lipid membrane, and that it contains a large number of specialised pigment molecules to harvest the solar energy, as well as small organic molecules to transport electrons through the complex. Unfortunately PSII is not a perfect machine; it sometimes breaks down, especially when the sunlight is very bright, and has to be repaired. To do this the damaged PSII complex is partially disassembled into a smaller complex, and the damaged protein is replaced by a newly made version. Without this special repair mechanism PSII would be quickly inactivated in the light and plant growth and oxygen evolution would be inhibited. The purpose of our research is to understand how PSII functions to split water, how PSII is assembled from its component parts and how it is repaired efficiently. Understanding these processes might allow us in the future to enhance photosynthesis in crop plants so that we can increase growth to help satisfy the ever increasing demand for more food and more biomass. This knowledge might also have applications in the design of new, sustainable herbicides or the design of new man-made catalysts that might act as 'artificial leaves' to provide renewable fuels from solar energy. Together with our collaborators, we have previously shown that PSII is assembled in a stepwise manner from smaller sub-complexes or modules and that these assembly intermediates also bind 'accessory factors' not found in the final active PSII complex. Whilst progress has been made in characterising the larger assembly intermediates, less is known about the early steps in PSII assembly. We propose to find out more about what is happening at this stage of PSII assembly. To do this we will employ a wide range of different experimental techniques and will work with collaborators around the world to maximise the return on the investment in time and money. By using a combination of genetic engineering and protein purification, we will isolate different types of early PSII assembly intermediate: some very early on in assembly when pigment molecules are first inserted into the protein as well as a minimal type of PSII complex, the PSII RC, that assembles later on in the pathway. By analysing the composition of these complexes we hope to identify new proteins that are required for assembly and repair of PSII. We have already identified two accessory proteins, termed Ycf48 and Ycf39, which bind to the PSII RC found in a cyanobacterium. We will employ a combination of mutagenesis, biochemistry and structural biology to find out more about their roles in PSII assembly, their 3D structure and how they interact with PSII. In addition we will use a special type of microscopy to detect the location of the PSII RC in live cyanobacterial cells to see where about this complex is found which will give us clues as to the site of assembly within the cell. Overall our research will provide important new information on how the oxygen-evolving complex of photosynthesis is assembled.

The oxygen-evolving photosystem II (PSII) complex is assembled via a set of distinct intermediates, some of which bind conserved 'accessory' proteins of unknown function. In this application we wish to clarify the subunit composition and activity of the so-called PSII reaction centre assembly intermediate (PSII RC) which binds the lumenal Ycf48 accessory factor. In background work to this proposal we have solved the structure of Ycf48 bound to a synthetic D1 C-terminal peptide by X-ray crystallography to 2.1Å. Objective 1 of the application will test the physiological relevance of this interaction using a site-directed mutagenesis approach in the cyanobacterium Synechocystis sp. PCC 6803. Objective 2 describes the use of His-tagging technology to isolate the PSII RC for detailed spectroscopic characterization. Mass spectrometry will be used to determine the subunit composition and to identify additional 'accessory' proteins. In background work to this proposal we have discovered that Ycf39 (slr0399), a member of the short-chain dehydrogenase/reductase superfamily, is also a component of the PSII RC. We will overexpress Ycf39 and its two close homologues (Slr0317 and Sll1218) in E. coli for structural studies and to test for NAD(P)H binding. Mutagenesis experiments will test for overlap of function between these homologues with regard PSII assembly and function. Objective 4 describes the use of confocal fluorescence microscopy to identify GFP-tagged PSII RC in live cells as a means of detecting the sites of PSII assembly in the cell. The precursor D1 subunit is inserted into the membrane via SynYidC (Slr1471). Other factors involved at this early stage of D1 insertion are unknown. Objective 5 describes a biochemical approach to identify these factors: flag-tagged or His-tagged derivatives of SynYidC will be isolated under conditions where the newly synthesised protein is jammed in the insertion apparatus and co-purifying proteins identified by mass spectrometry.

Understanding the details of the assembly of the oxygen-evolving complex of photosynthesis might in the long-term lead to the development of microalgae and plants with more robust and more efficient photosynthesis. In the agricultural sector, beneficiaries could include: companies involved in modifying or selecting plants to maintain and or improve crop yields under stress conditions either imposed by the changing environment or to exploit less fertile land; farmers who wish to develop new practices for similar reasons; governments and policy-makers interested in developing novel strategies to achieve food security; and the public who will benefit from food security. In the bioenergy sector, beneficiaries include: companies wishing to develop alternatives to fossil fuels; governments and policy makers who are interested in new routes to energy security and for new energy sources for developing countries; the military who are looking for alternative fuels for specific and niche uses; environmentalists who need to focus on rational long-term alternatives to fossil fuels. In the environmental and ecological sector, beneficiaries include those wishing to understand and mitigate loss of photosynthetic species or productivity in a changing environment. These include governments and policy makers, the tourism sector (coral reef bleaching) and fisheries (the loss of photosynthetic microbes at the start of the food chain).

Our research will also investigate at a fundamental level how proteins are inserted into the cyanobacterial thylakoid membrane and then assembled into multi-subunit complexes. Ultimately this knowledge will benefit those in the biotechnology sector who wish to develop cyanobacteria and other related photosynthetic organisms as solar biorefineries for the sustainable production of green chemicals and high-value products.

In the education sector, in museums and in the media, there will be benefits from publicising new advances in one of the most fundamental biological processes and one that has been taught at secondary school and so readily familiar to the general public.

Staff hired for the project will obtain training in cutting edge research in a world-leading research centre containing several world leading experts in Photosynthesis including Bill Rutherford, Jim Barber and Jasper van Thor as well as Peter Nixon and James Murray, with expertise ranging from femtosecond spectroscopy to biofuel production through metabolic engineering of photosynthetic microbes. They will have the benefit of the excellent intellectual environment of a leading university with a tradition of close ties with engineers and industry.