Quinone redox tuning for regulation and protection of the water splitting enzyme

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


Photosynthesis is the process that converts solar energy into the chemical energy that powers life. The light is used to split water, removing some of its electrons and using them to pull down carbon dioxide from the atmosphere to make the building blocks and fuel for life. When water is split in this way, protons (hydrogen ions) and oxygen are released. The oxygen accumulates in the atmosphere, reacting with UV to form the protective ozone layer. The oxygen also provides a reactive environment that allows respiration to occur. Both of these roles of oxygen were crucial for the development of multicellular organisms: life as we know it.
The most important photosynthetic enzyme is Photosystem II, the water splitting enzyme. It is the enzyme that changed the planet. Water is very unreactive and splitting it is hard to do. An enzyme capable of splitting water seems to have evolved only once and all O2-producing photosynthesizers, from the most ancient cyanobacterium to the oak tree, use the same enzyme.
Such difficult chemistry requires a lot of energy and this comes from sunlight. The amount of energy in light depends on its colour and Photosystem II uses red light absorbed by a pigment called chlorophyll a. The energy available in the light collected by chlorophyll is not enough to do what PSII does safely and although evolution has provided it with an impressive bag of chemical tricks designed to protect it from burning out, in the end it just takes the hit. It is destroyed after about a million reactions (about every half hour, depending on the brightness of the sunlight), and it then needs to be taken apart and the damaged subunits replaced with new ones. This damage and repair costs energy and under severe conditions it can limit plant growth and give smaller crop yields.
The present study is focused on discovering and understanding the tricks for protecting Photosystem II. We have previously found some interesting stuff. The damage occurs in PSII when the light is there, the system is ready to work but it can't do anything useful with the energy because something prevents the completion of the hot chemistry. When this happens the light-generated charges come back together again forming a high energy state of chlorophyll called a triplet. The triplet chlorophyll reacts with normal oxygen and turns it into a super-reactive form called singlet oxygen, which is the real killer. This causes the damage to Photosystem II.
In principle this damage could happen when electrons don't come from water, for example prior to the assembly of the water splitting catalyst, or when there is nowhere to put the electrons because of a downstream block, for example due to a lack of CO2 to fix. But in both of these cases burnout is minimised because a component called QA has its reactivity tuned down so that the energy is dumped as heat instead of doing the high energy reactions that form the triplet. When the water splitting part is assembled, or when the CO2 levels return to normal, QA is switched back to its high energy function.
We are now looking closely at how the next component in the chain, QB, works and if it too is tuned or controlled in a different way or indeed if it helps to tune its neighbour QA. Already we have had surprises and it seems QB works very differently from how some researchers thought.
By understanding the details of PSII damage and protection mechanisms, better strategies may be developed for making photosynthesis more efficient and increasing food production. Very recently other researchers got improved crop growth when they managed to accelerate (a different kind of) protective switching in plants. So this approach could just work.

Technical Summary

Our previous studies of QA, unearthed redox-controlled switching of backreaction pathways, and allowed us to deduce the mechanisms of photoprotection and photoinhibtion, which are at heart of photosynthetic bioenergetics. This project extends these studies to QB, the exchangeable quinone that is the carrier for electrons exiting PSII. The approaches used involve a range of biophysical and biochemical methods including electrochemistry (spectroelectrochemistry, redox potentiometry), spectroscopy (UV/vis absorption, fluorescence and EPR spectroscopies) plus luminescence and more. These methods will provide data ranging from thermodynamics, to electron transfer kinetics and rates of ROS production.
QB is relatively poorly studied despite its key role in PSII. We have taken up the challenge to attempt to fill the gaps in our knowledge. Our first results were remarkable: i) redox titrations were clear and directly contradicted the only (?) reported titration of QB and showed a situation totally different from expectations, with QB- being strongly stabilised thermodynamically and QB more tightly bound than QBH2; ii) our mechanistic studies on PsbS bound to PSII showed that PSII was inhibited at the level of QB, reminiscent of the inhibition seen prior to Mn-cluster assembly or when formate replaces bicarbonate as a ligand to the iron, and amazingly, the inhibition was reversed by bicarbonate addition. These two unexpected observations indicate that this QB project is not only going to be full of surprises and but also will provide key new mechanistic insights into PSII function. QB promises to be the place where the redox tuning that controls photoinhibition meets mechanistic control imposed by proton access, binding conformations and structural switches.
Enhancing the regulatory reactions of PSII can really improve crop yields. The current study promises to provide bioenergetics insights that could contribute to more efficient agriculture.

Planned Impact

Impact summary.
The proposed research falls under the remit of two BBSRC strategic priorities: "Bioenergy: generating new replacement fuels for a greener, sustainable future" and "Sustainably enhancing agricultural production". Central to both priorities is photosynthesis research and in particular research aimed at improving the energy efficiency of photosynthesis as both priorities rely on increases in crop yields.

1) The main outcome of the research is improving our understanding of the basic bioenergetics of Photosystem II, an enzyme central to life in that it is largely responsible for powering the biosphere and one with important applications, actual (e.g. all plant growth, effects on climate) and potential (as the bench mark enzyme for water oxidation in a world greatly in need of better water-splitting catalysts for solar fuel production).

2) The other major outcome is understanding how energy gaps between the electron acceptors are modulated (redox tuning) under a range of circumstances. This outcome includes: i) improved understanding of the role of this regulatory mechanism in photoprotection; ii) improved understanding of the mechanisms of ROS in photoinhibition; iii) understanding the consequences of the variation in energy gaps existing for the quinones in different species; iv) the demonstration that the regulatory protein, PsbS, has unexpected roles, not only in controlling electron transfer at the level of the quinones, but also most likely in redox tuning for protecting PSII.

The main beneficiaries of this research are listed below.

Academic and education sector. The output of the proposed research will bring new insights for understanding the basic bioenergetics of PSII and the regulatory mechanisms in photosystem II, the water oxidising enzyme. The enzyme is at the heart of energy conversion and responsible for making the planet aerobic. It thus features in most biology courses. Any advances in the basic energetics should have a major impact academically not only in the field and but also for non-specialists, students and writers of text books. This is potentially text book stuff and thus could impact the education sector. The possibility of new regulatory mechanisms in crops interests the academic sector and brings a new world of potential biotechnology applications.
Biotechnology and agricultural sector. Studies on regulatory mechanisms affecting photosynthetic efficiency are of potential relevance to the great problems of the sustainability of agriculture and biotechnology. Improved biomass production was recently achieved by tuning the regulatory response of the non-photochemical quenching apparatus via genetic engineering of maize (Kromdijk et al. Science 2016 354: 857-861). The outcomes of this research (point 2 above) could provide new strategies to allow these regulatory mechanisms to be used to obtain the improved efficiencies. It will also help to understand the influence of the redox tuning on herbicide binding.
Policy makers, environmental, ecological, agricultural sectors: The outcomes of point 2 could allow information-based judgements on the feasibility of improved crop yields for food and energy. The information needed will be provided to policy makers in government, to research councils, and to groups interested in ecological questions and sustainability.

Press and public Topics associated with agriculture productivity and food security are certain to attract the attention of the press and the public. The outcomes in part 2 above, will most certainly be of interest to these sectors.


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De Causmaecker S (2019) Energetics of the exchangeable quinone, Q, in Photosystem II. in Proceedings of the National Academy of Sciences of the United States of America

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Kornienko N (2018) Oxygenic Photoreactivity in Photosystem II Studied by Rotating Ring Disk Electrochemistry. in Journal of the American Chemical Society

Description We have measured the key thermodynamic properties of the second electron acceptor quinone, QB, in photosystem II. This is the terminal electron acceptor within the reaction centre and as such is important in controlling electron flow at the start of the photosynthetic electron transfer chain, It is also the target of many important herbicides. The results change the picture that exists in the literature. The existing work is directly contradicted by our direct measurement of the 1 electron reduced state as a thermodynamically stable state. This break though changes our view of bioenergetics mechanism of this key cofactor. It's publication is bogged down by having to determine what went wrong in the earlier eroneous study. We hope it will be published soon. We have several other good results that are piling up on this project and hope to wrap them up when the major findings are published. The psbS study is nearly complete and is in a near final draft. A study of modulation of semiquinone reactivity with O2 is also nearly complete. A third study on the kinetics of electron transfer through the quinones advanced through a collaborative visit to use custom built spectrophotometer in Paris. This study too has advanced well and needs to written up.
Exploitation Route The results will be relevant to
1) herbicide action,
2) understanding regulation and protection in photosynthesis which has been demonstrated to be affect crop yields.
3) the bioenergetics mechanism of QB should update understanding of this important enzyme and we expect this to feature in textbooks.
Sectors Agriculture, Food and Drink,Education,Energy

Description Reisner Cambridge 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution I began studies of PSII on electrodes in 2008. I initiated a collaboration with Erwin Reisner when he first worked in Manchester suggesting that we use my water oxidising biohybrid cell TiO2 +PSII in conjuction with his O2 tolerant H2ase to make a water splitting/H2 prodcuing cell. We provide the expertise on PSII , how it works, the Three papers came out of this study culminating with the planned cell. The first two were developmental papers establishing the ground work and methods. The papers were jointly written.
Collaborator Contribution The Reisner group introduced the use of meso ITO materials, controlled orientation of the PSII and completed the system with their H2ase.
Impact Three joint research articles came out of this work.
Start Year 2010
Description kreiger ROS studies Saclay 
Organisation Saclay Nuclear Research Centre
Country France 
Sector Public 
PI Contribution We discovered a new regulatory effect in photosynthesis. We predicted that this would lead to increased formation of reactive oxygen species. So we contacted my ex-colleague in Saclay, Anja Keiger Liszkay, and invited her to make test this prediction.
Collaborator Contribution Anja Kreiger Lizskay measured the concentration of singlet oxygen generated in PSII in the absence and presence of bicarbonate using spin trapping EPR. She became interested in discovery and interpretation that we made and has gone on to test these ideas using mutant plants in her lab.
Impact Published article in Proc Nat Acad Sci USA in October 2016. This article was seen as a big breakthrough and has already led to a full article discussing it in Trends in Plant Sciences. We hope to write up the follow on this subject when it is completed.
Start Year 2015
Description 1 talk at junior scientist pre-congress meeting (Sven De Caussmaecker) and 2 posters (Sven de Causmaecker and Andrea Fantuzzi) at European Photosynthesis Congress Uppsala June 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Post Doc Sven De Causmaecker gave a selected talk on QB bioenergetics in PSII at the pre Conference junior scientists meeting and presented a poster at the congress. Andrea Fantuzzi presented a poster on semiquinone reactivity in PSII. The subject was the redox potential of the secondary quinone.
Year(s) Of Engagement Activity 2018
Description Bioenergetics Christmas Meeting 2017 A.W. Rutherford Plenary lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Rutherford gave the plenary lecture at the annual bioenergetics meeting of the Biochemical Society presenting work that came from research done under the the three BBSRC grants below: photoactivation, nitroplast and far red light
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/PreviouslySupportedEvents/tabid/1202/ModuleId/6547/View/Conferen...