Role of Atypical D1 Proteins in Photosystem II

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Murray J (2020) Photosystem II in a State of Disassembly in Joule

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Bec Ková M (2017) Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Yu J (2018) Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Our outputs are delayed because of COVID, but one result is that only a small mutation in a key photosystem gene is sufficient to enable green organisms to convert the usual chlorophyll a to chlorophyll f, the far-red chlorophyll. This result has potential applications to growing plants more efficiently in lower energy near-infrared light. Nature Plants paper https://doi.org/10.1038/s41477-020-0616-4
Exploitation Route The development of new crops with engineered light harvesting systems.
Sectors Agriculture, Food and Drink,Energy

 
Description Chlorophyll-f-containing Photosystem I
Amount £723,298 (GBP)
Funding ID BB/V002015/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2021 
End 12/2023
 
Description Probing the structure and function of a super-rogue photosystem II complex involved in chlorophyll f synthesis
Amount £594,088 (GBP)
Funding ID BB/V002007/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2021 
End 09/2024
 
Description Algaerium Bioprinter and Algae Printing, STARTS Prize '17 Exhibition, Ars Electronica Festival, Post City, Linz, Austria: Marin Sawa 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Marin Sawa, designer in residence in the Nixon group, presented her work on the use of the algaerium printer to print algae. The exhibit alerted the audience to current biotechnology uses of algae and the importance of photosynthesis in general. The activity was also publicized in the Phyconet newsletter in September 2017 https://mailchi.mp/cb67160f97d6/w7vlidhdnp-1081961?e=e8c903b6d7
Year(s) Of Engagement Activity 2017
URL https://starts-prize.aec.at/en/algae-printing/
 
Description Algaerium Bioprinter and Algae Printing, STARTS Prize '17 Exhibition, the BOZAR Electronic Art Festival, Centre for Fine Arts, Brussels, Belgium: Marin Sawa 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Marin Sawa, designer in residence, presented her work on algae printing at an International Arts Festival. The work highlights the biotechnology applications of algae and cyanobacteria and the importance of photosynthesis in general.
Year(s) Of Engagement Activity 2017
URL https://starts-prize.aec.at/en/algae-printing/
 
Description Annual debate at the Linnean Society of London, Central London, on 'Plant Biology and the Future' 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Gave short presentation about my research with emphasis on the potential impact for society in terms of breeding better crops and developing solar biorefineries for biotechnology applications. Member of panel that debated the future of plant science.
Year(s) Of Engagement Activity 2019
URL https://www.linnean.org/meetings-and-events/events/annual-debate-the-future-of-plant-science
 
Description Green Great Britain Week, Imperial Lates 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Exhibit on the 'solar bio-battery', a printed biophotovoltaic cell consisting of cyanobacteria grown on an electrode which converts sunlight into an electric current
Year(s) Of Engagement Activity 2018
URL https://www.imperial.ac.uk/news/188694/imperial-lates-launches-with-evening-greener/
 
Description Imperial College outreach event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Ran a stand with explanation of oxygen evolution of photosynthesis, and samples of pond weed releasing oxygen.
Year(s) Of Engagement Activity 2017
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/eventssummary/event_12-10-2017-15-53-1...
 
Description Imperial Fringe, walking in the air 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A demonstration of photosynthesis to visitors to Imperial College
Year(s) Of Engagement Activity 2017
URL https://www.facebook.com/imperialcollegelondon/videos/10155851044071838/?hc_ref=ARTULrU74j0rtS62N5zy...
 
Description School Visit (Watford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk to a 6th form students on my scientific work.
Year(s) Of Engagement Activity 2019
URL https://www.watfordboys.org/
 
Description Solar wallpaper, Imperial Fringe: Walking in the Air, Main Entrance, South Kensington Campus, Imperial College London : Marin Sawa 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Marin Sawa, designer in residence, presented an exhibit highlighting the potential use of printed algae in the form of a wallpaper to cleanse air in the indoor environment. Her work demonstrated to the audience some of the fascinating ways that algae can be used in the biotechnology area as well as the importance of photosynthesis in general.
Year(s) Of Engagement Activity 2017
URL https://www.facebook.com/imperialcollegelondon/videos/10155851044071838/?hc_ref=ARTULrU74j0rtS62N5zy...
 
Description short talk at Linnean Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A debate on the future of plant science in the Linnean Society, with questions and discussion afterwards.
Year(s) Of Engagement Activity 2019
URL https://www.linnean.org/meetings-and-events/events/annual-debate-the-future-of-plant-science