Award details

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

Reference	BB/R00921X/1
Principal Investigator / Supervisor	Professor Alfred Rutherford
Co-Investigators / Co-Supervisors	Dr Andrea Fantuzzi
Institution	Imperial College London
Department	Life Sciences
Funding type	Research
Value (£)	641,891
Status	Completed
Type	Research Grant
Start date	01/03/2018
End date	28/02/2022
Duration	48 months

Abstract

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.

Summary

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 lightis 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.

Impact Summary

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 impactacademically 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.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
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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