Award details

Role of Atypical D1 Proteins in Photosystem II

Reference	BB/P00931X/1
Principal Investigator / Supervisor	Dr James Murray
Co-Investigators / Co-Supervisors	Professor Peter Nixon, Professor Alfred Rutherford
Institution	Imperial College London
Department	Life Sciences
Funding type	Research
Value (£)	584,482
Status	Completed
Type	Research Grant
Start date	01/04/2017
End date	30/09/2020
Duration	42 months

Abstract

The oxygen-evolving complex (OEC) in PSII is located at a site with highly conserved amino acids, mainly binding carboxylate groups from the D1 protein. This complex is responsible for the water oxidation reaction that generates nearly all of the oxygen in the atmosphere. We have identified a class of PSII in which this consensus is modified, such that residues known to be essential for oxygen evolution are mutated. These are termed the "rogue", rD1. The rD1 are phylogenetically old and are present mainly in diazotrophic cyanobacteria. The nitrogenase enzyme is irreversibly inhibited by oxygen. We predict that the rogue D1 are capable of forming a structural PSII complex that is not capable of oxygen evolution. It is however possible that rD1 forms a new kind of reaction centre, with a different electron donor to PSII. There is another class of even more different D1, called srD1, which are even older than rD1, and are associated with far red light adaptation and the production of chlorophyll f. To investigate the rD1 and srD1 sequences we will insert them into the chromosome of an appropriate strain D1 triple-deletion strain of Synechocystis PCC 6803. The resultant chimeric complexes will be purified and analysed biochemically and biophysically, e.g. by oxygen evolution and elemental analysis for metal content, along with spectroscopy, fluorescence, thermoluminescence and EPR measurements. We will later purify tagged rD1-PSII complexes from the host organisms to generate near-wild type complexes for characterization. We will also examine the rD1 and srD1 in vivo, following their expression under different conditions, and correlating it with factors like oxygen evolution and nitrogen fixation ability. These two strands in vivo studies and characterization of purified complexes should tell us what the rD1 and srD1 are doing and how.

Summary

In photosynthesis, light is used to remove the hydrogen from water to give oxygen. The hydrogen equivalents are then used to chemically reduce carbon dioxide to organic molecules. The water oxidation reaction is catalysed by an enzyme known as photosystem II (PSII). It is hard to catalyse this reaction with light, so far the only known effective system is the natural one. The reaction takes place at a metallocluster containing 4 manganese ions and a calcium ion. This cluster is bound and stabilised by a highly conserved protein known as the "D1 protein" in PSII. The PSII complex contains more than 20 other protein subunits. However, in an active leaf, the D1 protein is degraded extremely quickly, every 30 minutes or so. This is thought to be because of the generation of reactive oxygen side products from the water-splitting reaction which damage the protein. A sophisticated system of repair exists to regenerate PSII with fresh D1 protein. Cyanobacteria have several different D1 genes suited to different situations. Some are synthesized in response to high light, others in low oxygen environments. Some photosynthetic cyanobacteria can also fix nitrogen from the air. This can be a problem as the nitrogenase enzyme is irreversibly inhibited by oxygen. These organisms either physically separate the nitrogenase from photosynthesis, or only fix nitrogen at night when PSII is inactive. There is a recently discovered a class of D1 genes that are mutated relative to the "canonical D1" at the sites binding the manganese cluster. The mutations are such that these "rogue" D1 are not thought to be capable of oxygen evolution, however, there are sufficient functional groups for metal binding to be a possibility. We believe that the atypical D1 sequences might be a mechanism to inactivate PSII with a non-catalytic D1, which is then replaced when activity is required again. There is a further class of even more atypical D1 ("super-rogue D1") that is associated with cyanobacteria that adapt to far-red light by making a red-shifted chlorophyll, chlorophyll f. This chlorophyll has a peak absorption in the infra-red, yet seems to still be capable of using these lower energy photons to drive water oxidation. The super-rogue D1 is 1000-fold up-regulated in far-red light conditions, so probably has a role in adaptation to far-red light. The atypical D1 sequences are phylogenetically early, so are reminiscent of an ancestral D1, so could provide information on the evolution of oxygenic photosynthesis. If functional in substrate oxidation, the variant PSII may use a substrate other than water. Of interest in itself and again providing insight into the evolution of photosynthesis. Such a reaction centre would be a novel finding. We will investigate the function of the atypical D1 proteins in PSII both in vivo and in vitro. For in vivo studies we will culture cyanobacteria with atypical D1 under a variety of conditions, including circadian light-dark rhythms. We will investigate the expression pattern of the atypical D1 in comparison to normal D1, in relation to other factors, such as light-dark, nitrogen fixation, and external carbon sources. For in vitro studies we will purify PSII with the rogue and super-rogue D1. We will investigate their function, such as metal content, ability to oxidise substrates and transfer electrons. We will assess the presence of all of the PSII subunits in the modified reaction centres. With a combination of the in vivo and in vitro approaches we will learn what the biological function of the atypical D1 sequences is, and how, at a biochemical and biophysical level, it is accomplished.

Impact Summary

The global energy consumption rate at present is approaching 16 TW and will rise towards 20 TW within this decade. The energy provided by solar radiation is equivalent to 100,000 TW. That is, more solar energy strikes the surface of the earth in an hour than all the global fossil energy consumption in an entire year. About 3 billion years ago, living organisms developed molecular mechanisms to take advantage of this vast energy resource and it was their photosynthetic activity which allowed life on our planet to prosper and diversify on an enormous scale. Any improvement in our understanding of any single facet of photosynthesis may immediately have far-reaching economic implications both for agriculture and biomimetic artificial photosynthesis. For solar radiation to be utilized on a massive scale, while at the same time minimising the interception area and not competing with food production, efficiencies greater than that achieved by biomass production are required. In terms of solar energy conversion, the early stages of photosynthesis, including the water splitting reaction, are highly efficient, while the production of biomass is less so. For this reason it is important to understand the molecular details of the highly efficient energy conversion reactions that occur before the fixation of carbon dioxide (i.e. light reactions). Understanding the details of the rogue D1 protein of PSII might in the long-term lead to the development of cyanobacteria with more robust and efficient photosynthesis. In the ocean, cyanobacteria such as the rD1-containing Crocosphaera are major nitrogen fixers, and play a large role in global nitrogen cycling. This work will give insight into the cycling of nitrogen and oxygen in the ocean of interest to microbiologists, oceanographers and geochemists. 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 techniques and research in a world-leading research centre. The photosynthesis groups at Imperial College have expertise ranging from cyanobacterial microbiology to femtosecond spectroscopy. The PDRA and technical staff will be in an excellent position to progress their careers. They will have the benefit of the excellent intellectual environment of a leading university with a tradition of close ties with engineers and industry.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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