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, QB, in Photosystem II. in Proceedings of the National Academy of Sciences of the United States of America

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Fantuzzi A (2022) Bicarbonate-controlled reduction of oxygen by the QA semiquinone in Photosystem II in membranes. 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

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Zamzam N (2020) Femtosecond visible transient absorption spectroscopy of chlorophyll- f -containing photosystem II in Proceedings of the National Academy of Sciences

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 providing a clear and intuitive understanding of the thermodynamics which explains the mechanism of the quinone reduction function of PSII. The existing literature report no stable semiquinone, is directly contradicted by our direct measurement of the 1-electron reduced state as a thermodynamically stable state. This breakthough changes our view of the bioenergetics mechanism of this key cofactor. It's publication was bogged down by having to determine what went wrong in the earlier erroneous study. It is now published in PNAS.
We have several other good results that are piling up on this project drafts of manuscripts have been written and are currently being finalised and some published.
a) The psbS study is published as a preprint but has advanced slowly because the international collaboration and the pandemic..
b) A study of modulation of semiquinone reactivity with O2 is now published in PNAS. Our lengthy experimental study are completed was complimented by the inclusion of advanced computation calculations by our collaborator Ville Kaila which backed up our interpretation of O2 binding directly to the non-heme iron, This article was held up by at least 18 months due to the pandemic.
c) A study on the kinetics of electron transfer through the quinones advanced through a collaborative visit to use custom built spectrophotometer in Paris. This has been held up because the post doc doing the study left, the arrival of the new post doc was blocked by the pandemic and when he arrived he was prevented by travel restrictions to travel to Paris because of the pandemic. This part of the project is on hold.
d) We have collaborated to measure CO2 release from PSII, differentiating between CO2 release associated with bicarbonate-enhanced proton release on the electron donor side and the bicarbonate release on the electron acceptors side. This has been published but the pandemic reduced our options to do further experiments to make the results irrefutable. We have an alternative approach that we hope to work on when the COVID restrictions are reduced.
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.
4) In collaboration with Erwin Reisner's group we introduced the use of the rotating ring disc electrode into photosynthesis research
Sectors Agriculture, Food and Drink,Education,Energy

URL http://www.imperial.ac.uk/news/192950/sustainable-growth-energy-insights-news-from/
Description Alain Boussac: Collaboration with CNRS CEA Saclay France 
Organisation Saclay Nuclear Research Centre
Country France 
Sector Public 
PI Contribution Two studies: 1) I provided some some input a good deal of the basic thinking that set the stage for the research. I provided some of framework for the interpretation and the impetus for calculating the overall bioenergetic scheme. I also helped with the interpretation of several aspects. 2) I contributed to the interpretation on an in depth EPR study of the Mn in PSII. This work is a continutation of work that I initated when I was head of this group in France. Some of the EPR phenomena were my own discoveries from my work in the 1980's.
Collaborator Contribution 1) Alain Boussac did the EPR studies, Miwa Sugiura's group made the mutants, Fabrice Rappaport did the UV vis studies and the calculation 2) Alain Boussac performed the EPR experiments, developed the biochemical treatments applied and did the the first level of interpretation.
Impact Two research articles were published in these collaborations. This work is at the boundry of biology chemistry and physics. Further collaborations are under way.
Start Year 2013
Description Kaila DFT of PSII 
Organisation Technical University of Munich
Country Germany 
Sector Academic/University 
PI Contribution I helped to initiate and focus advanced dft calculations on specific reactions occuring in water splitting enzyme and I helped to interpret the findings and write the article.
Collaborator Contribution Dr Ville Kaila and his student performed the dft calculations and the main interpretation of the findings.
Impact An article was published in 2016 which has an important impact on the field. I associated Prof Kailla in an on-going collaboration with Alain Boussac, Johannes Messinger on spin state changes and pH in the S2 to S3 transition and this has provided useful insights to that study. A meeting was held here at Imperial by all 4 groups in January and a paper is being drafted based on the outcome of this interdisciplinary study. Interdisciplinary: Biochemistry, Physical Chemistry, Computational Chemistry, Biophysics, Spectroscopy, Molecular enzymology,
Start Year 2014
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 Sugiura 
Organisation Ehime University
Country Japan 
Sector Academic/University 
PI Contribution This is a long running but sporadic collaboration in which Miwa Sugiura has developed several mutated or engineered starins of an thermophilic cyanobateria. The first of these were done in my lab in France 15 years ago. In the period covered by this grant the collaboration involved charaterisation of a mutant which had been made specifically to change the protential of the pheophytin cofactor with predicted effects on kinetics and ROS production. My role was to provide some of the theoretical background but mainly to be involved with the development of a kinetic calultion based on the redox potentials and the distances in order to verify my original hypothesis on how redox tuning influences these processes.
Collaborator Contribution Miwa Sugiura mde the mutants and isolated the enzyme as well as doing several experiments on the susceptibility of the cells to photodamage etc.
Impact Two papers published as coauthors.
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...
Description European Bioenergetics Conference August 2022 Aix en Provence France Plenary Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary Lecture on Photosystem II Bioenergetics
Year(s) Of Engagement Activity 2022
Description News release on College Website upon publication of the PNAS 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact students interested in energy and sustainability were made aware of the energy accounting research occurring at imperial college
Year(s) Of Engagement Activity 2019
URL https://www.imperial.ac.uk/news/192950/sustainable-growth-energy-insights-news-from/
Description Plenary lecture French Society of Bioenergetics (Autrans) 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary lecture on bioenergetics at an international meeting organised by the French Bioenergtics Society
Year(s) Of Engagement Activity 2019