Photosynthetic water oxidation driven by near infra-red light

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

Abstract

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 (PSII), 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 PSII uses red light absorbed by a pigment called chlorophyll a. Until recently it was thought that all PSIIs have chlorophyll a at the heart of the process. There have been decades of discussion about why red light (680nm) is the lowest energy needed to perform water oxidation: this is known as the red-limit.
The red limit was questioned when it was found that a marine bacterium, which was shaded by a green sea-squirt (!), had chlorophyll d performing the photochemistry at around 710nm. An even longer wavelength pigment, chlorophyll f, was discovered recently. This time it was not just a quirky one-off in a weird ecological niche, chlorophyll f was found to be present in a wide range of common cyanobacteria. However the chlorophyll f is only made when they grow in near-darkness, shaded from visible light but exposed to far-red/near-IR light, e.g. deep in bacterial mats in hot springs, or in some rocks. The role of chlorophyll f is generally considered to be only for gathering light but not for the photochemical part of photosynthesis. We have now found that the chlorophyll f does seem to perform photochemistry in PSII. This surprising result represents a major extension of the red limit.
These strange far-red PSIIs perform normal PSII chemistry and yet they are quite different from normal PSII in energy terms. In the present project we intend to study this new world of long-wavelength photosynthesis, to follow up our surprising discovery, to understand how it works, to assess what changes have occurred that allow PSII to function with less energy, and to see if the move to lower energy gives better energy efficiency. Since it seems unlikely that there is such a thing as a free lunch, we shall also test if the improved energy efficiency comes with penalties in terms of it resilience to variations in light intensity, for example. This project will involve studying PSII in living cells, membranes and in the isolated enzyme using a range of biochemical and biophysical methods.
This demonstration of oxygenic photosynthesis working well beyond the established red limit, takes us into a realm of the subject that is largely unstudied; and yet longer wavelength photosynthesis is already a high profile engineering target aimed at making crops and bioenergy more efficient. Normal photosynthesis is inefficient and much effort goes into thinking up ways of improving it. Engineering longer wavelength photosynthesis seemed a far-off pipedream but now it turns out that nature has already done the engineering. Our aim here is to determine if moving to far-red photosynthesis will provide a useful technological target with an improved energy budget and to test if it comes with a loss of resilience that could restrict the use of engineered long-wavelength photosynthesis to specific growth conditions.

Technical Summary

The energy needed for PSII comes from the light absorbed by chlorophyll a (Chl a) at 680nm, 1.82 eV. This energy is accounted for by the energy trapped by water oxidation, quinone reduction and PMF formation, and by the energy lost as heat to ensure a high quantum yield of charge separation, stability of the redox intermediates and an adequate driving force for catalysis and product release. We have shown that this energy is too low to avoid damaging back reactions and this is a major contributor to photoinhibition. This "energy squeeze" on PSII explains many of its properties including the multiple cases of redox tuning that protect it against back-reactions.
The recent discovery that some cyanobacteria use the far-red pigment Chl f, and our new observations that Chl f is involved in charge separation, allow PSII to be studied when the energy available is decreased by >100meV.
We shall analyze Chl f-driven PSII function using biophysical methods: measuring absorption, action and fluorescence spectra, quantum yields of charge separation and water oxidation, luminescence yields, redox potentials and temperature effects. This should clarify the basic bioenergetics, showing which energy gaps decrease to account for the lower energy input.
We shall also study photoinhibition in far-red PSII in high and variable light, testing our predictions of a resilience penalty. The results will be relevant to proposals to engineer long wavelength photosystems to improve the energy efficiency of agriculture and biotechnology.
These studies will be done on living cells, isolated membranes and isolated enzymes.
We shall also characterize far-red PSII biochemically, isolating and purifying PSII, analyzing the pigment content, the protein variants present, and developing molecular biological methods in the Chl f-containing strains. This will allow tagging of the proteins for scaling up purification and will also allow mutagenesis studies to test pigment assignments.

Planned Impact

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 to improve our understanding of the bioenergetics of Photosystem II, an enzyme central to life and one with important applications, actual (e.g. all plant growth, maintaining the 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 second outcome is to analyse far-red photosynthesis, a currently much publicised bioengineering goal aimed at improving sustainability. The research outcomes will be relevant to this approach and could determine 1) whether it is feasible, 2) if feasible, how to bring it about in engineering terms, and 3) how best to implement it in terms of the conditions in which it could be beneficial, e.g. in bioreactors/greenhouses vs in the open.

The main beneficiaries of this research are listed below.

Academic and education sector.
The output of the proposed research will bring unique insights to the understanding of the basics of energy conversion in photosystem II, the water oxidising enzyme, which is the most important of the light-converting enzymes in biology. It will test our current understanding by providing a version of the enzyme that does the same job chemically but does it with less energy. Understanding this will have major impact academically in the field and for non-specialists interested in bioenergetics. This is potentially text book stuff and thus could impact the education sector.
The possibility of long wavelength crops interests the academic sector and brings a new world of interesting bioenergetics problems.
Academic researchers in artificial photosynthesis will be interested in the energy limitations for biological water splitting. It seems likely that similar limitations may be relevant to the same chemistry done without a protein environment. Artificial photosynthesis is not yet a viable technology but it is moving rapidly. Future impact statements may see this section moved into the industrial impact category.

Biotechnology and agricultural sector.
All studies on the energy balances in agriculture are of potential relevance to the great problems of the sustainability of agriculture and biotechnology. Improved understanding of the bioenergetics of normal photosynthesis will already bring insights that could be directly useful to the sector. The specific questions answered in this research (point 2 above) could determine the implementation of long wavelength photosynthesis, whether it should be done, in what way and under what conditions, in order to obtain the improved efficiencies.
Policy makers, environmental, ecological, agricultural sectors:
The outcomes in point 2 will have knock-on effects on feasibility and implementation of this approach, and these will impact policy makers in government, research councils and groups interested in ecological questions and sustainability.

Press and public
Improving photosynthesis by moving to long wavelengths has caught the imagination of the press and the public. The outcomes in part 2 above, whether in favour or against this technology should remain of interest to these sectors.

Publications

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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 of the United States of America

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Zamzam N (2019) Femtosecond infrared spectroscopy of chlorophyll f-containing photosystem I. in Physical chemistry chemical physics : PCCP

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N├╝rnberg DJ (2018) Photochemistry beyond the red limit in chlorophyll f-containing photosystems. in Science (New York, N.Y.)

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Judd M (2020) The primary donor of far-red photosystem II: ChlD1 or PD2? in Biochimica et biophysica acta. Bioenergetics

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Antonaru LA (2020) Global distribution of a chlorophyll f cyanobacterial marker. in The ISME journal

 
Description Photochemistry in Photosystem II (PSII), the water/plastoquinone photo-oxido reductase, occurs from the first excited state of a chlorophyll-a molecule, absorbing red light at 680 nm. PSII exhibits several instances of redox tuning, all apparently aimed at protecting against photodamage, and indicating that its efficiency is limited by the energy available in the red-light [1]. This "energy squeeze" is considered to define the energy limit ("red limit") for oxygenic photosynthesis [1]. However, this view is questioned by the existence of two different, low-energy, long-wavelength (720nm) PSII paradigms [2]. The chlorophyll-d-PSII from an epiphytic cyanobacterium, Acaryochloris marina, has all but 1 of its 35 chlorophyll-a molecules replaced by chlorophyll-d (see [2,3] and references therein). By contrast, the more recently discovered, facultative far-red PSII, has only 5 far-red pigments (1 chlorophyll-d and 4 chlorophylls-f) but maintains 30 chlorophyll-a [2]. The primary electron donor at 720 nm was identified as the monomeric chlorophyll, known as Chl-D1 [2]. Three longer-wavelength chlorophylls-f collect light and pass excitation energy up-hill to the primary donor. Comparisons of standard chlorophyll-a-PSII with the two long-wavelength paradigms provide several new insights [2,3] on, e.g., charge separation and excitation transfer, photosystem evolution, and energy limits and efficiency. Chlorophyll-d-PSII and chlorophyll-f-PSII have evolved different strategies to work in far-red light and are impacted differently by the energy shortfall. Redox tuning of the electron transfer cofactors and the layout of the far-red pigments determine the trade-off between efficiency and resilience. Photosynthesis, it seems, can break the red limit but not the "law" that states there is no such thing as a free lunch.


[1]. A.W. Rutherford, A. Osyczka, F. Rappaport, Back-reactions, short-circuits, leaks and other energy wasteful reactions in biological electron transfer: Redox tuning to survive life in O2. FEBS Lett. 586 (2012) 603-616.

[2]. D.J. Nürnberg, J. Morton, S. Santabarbara S., A.Telfer, P. Joliot, L.A. Antonaru, A.V. Ruban, T. Cardona, E. Krausz, A. Boussac, A. Fantuzzi, A.W. Rutherford, Photochemistry beyond the red limit in chlorophyll f-containing photosystems Science 360, (2018) 1210-1213

[3] S. Viola, W. Roseby, S. Santabarbara, D. Nürnberg, R. Assunção, H. Dau, J. Sellés, A. Boussac, A. Fantuzzi, A. W. Rutherford, Impact of energy limitations on function and resilience in long-wavelength Photosystem II eLife (2022)11:e79890. https://doi.org/10.7554/eLife.79890
Exploitation Route Our discovery that the chlorophyll f-containing photosystems use a long wavelength chlorophyll for the primary electron donor was a suprise to the field, as it had been generally accepted that the long wavelength chlorophyls were simply light collectors, not photochemically active. This kind of reaction centre was descibed by a reviewer and editor as a new paradign in the field. The discovery that the absorption spectra of the 5 long wavelength pigments including the primary donor, were separated ifrom each other as well as from the chlorophyll a manifold, was an undreamt of situation in which specific chlorophylls could be studied without the need for tortuous and ambiguous deconvolutions, typical of the studies of the standard chlorophyll a containing photosystems. The new chlorophyll-f paradigm, brought new insights to the canonical chlorophyll a PSII and tp the other long wavelength PSII, the near-all chlorophyll-d containing PSII of Acaryochloris marina. The opened a new area of research in the field with many new projects focussing on this new experimental system.
Sectors Agriculture

Food and Drink

Education

Energy

Environment

Manufacturing

including Industrial Biotechology

 
Description The Science article (Nurnberg et al 2018) had a significant impact it was picked up by more than 90 news outlets. It also ran on several international radio stations. It was recommended by F1000 prime. It was the most viewed news story from Imperial College in 2018 and members of the Chinese academy of science and the Chinese academy of engineering ranked it as the 2nd most important advance in 2018 (after the Mars landing). The PI did 2 radio interviews and discussed with several science jurnalists who wrote articles on the subject. Some of the co-authors also did interviews and provided information for articles in the press. The breakthrough by our team initiated a new subject of study in the field, with many researchers turning their attention to this field.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Education,Energy
Impact Types Cultural

Societal

Economic

Policy & public services

 
Description Policy work committees
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
URL https://royalsociety.org/topics-policy/projects/low-carbon-energy-programme/net-zero-aviation-fuels/
 
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 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 ultrafast spectroscopy van Thor lab imperial 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution In our program to study far-red, chlorophyll f containing photosystems we wished to understand the photochemical reaction using time resolved spectroscopy. My group discovered that these systems do photochemistry at long wavelengths. We developed the isolation procedures, and generated functional models from a wide range of spectroscopy. We supplied the samples and worked with the van Thor group, developing the experimental conditions and some new spectrophotometers. We played important roles in experimental design and interpretation of the data as well as wring the manuscript.
Collaborator Contribution The van Thor group designed and built the ultrafast set-ups and did the data treatment. They also contributed to the interpretation and the write up.
Impact Multi-disciplinary: biochemistry, biophysics, physical chemistry
Start Year 2016
 
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 A Fantuzzi invited research seminar at Dept Chem TU Munich Dec 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Andrea Fantuzzi visited the lab of our collaborator Prof Ville Kaila in Munich. He spent 2-3 days discussing the on-going computational chemistry project providing detailed input and insights. He also gave a research seminar on the subject of far-red light photosynthesis to the Chemistry Department.
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 Dennis Nurneburg (post doc) and Laura Antonaru (PhD student) each prosented posters at 2 meeting in Vancouver 1) Photosynhetic Prokaryotes meeting 2) Int Soc Photosynth Res meeting. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The far red light work (Nurnberg et al 2018 Science) was presented to the US photosynthesis community by Dennis Nurnberg, the post doc employed on the grant.
Laura Antonaru present her work in which she has developed a bioinformatics approach to find new strains of far-red cyanobacteria.
Year(s) Of Engagement Activity 2018
 
Description European Bioenergetics Conference 2022 Aix en Provence France invited talk (Stefania Viola) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk on long wavelength photosynthesis
Year(s) Of Engagement Activity 2022
 
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 Gordon Conference invited talk Main USA 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited research talk in the main research meeting photosynthesis biophysics
Year(s) Of Engagement Activity 2019
 
Description International Bioenergetics Meeting at Imperial College (Bunty Meeting) Dec 8th 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Unusually this was a second of these meeting in 2017. Rutherford played a role in providing a research talk as did Nurnberg. The research was related to both the previous photoactivation grant, the nitroplast grant and the new far red grant. Rutherford also played the role of discussion leader and session chair.
Year(s) Of Engagement Activity 2017
 
Description International Conference on Porphyrins and Phthalocyanines ICPP-12 madrid July 2023 (Keynote Lecture) A W Rutherford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote lecture given of chlorophyll f containing Photosystem II
Year(s) Of Engagement Activity 2022
 
Description Invited lecture from the Students Biochemical Society of Impreial College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact The student Biochemical society occasionally invite an academic to give a lecture, They were interested in the far -red light paper that had come out in Science amit the big media splash.
Year(s) Of Engagement Activity 2018
 
Description Invited talk at Bioenergergetic discussion meeting at Imperial College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A. W. Rutherford gave a talk and chaired the discussion of the first session at the annual International Bioenergetics discussion meeting at Imperial
Year(s) Of Engagement Activity 2018
 
Description approx 90 print and on line articles, main stream newspapers and magazines as well popular sciences magazines, associated with our Science article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact several interviews with international journalists on our article in Science

https://www.thehindu.com/sci-tech/science/new-type-of-photosynthesis-discovered/article24203547.ece
https://www.newsweek.com/scientists-discover-new-photosynthesis-alien-life-982782
Year(s) Of Engagement Activity 2018
URL https://www.pourlascience.fr/sd/biochimie/la-photosynthese-fonctionne-aussi-dans-linfrarouge-14047.p...
 
Description press release Imperial college 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact press release on the research published in Science on far red photosynthesis.
Intended purpose to reach other media.
The press release was had the highest number of read for 2018 of all Imperial College press releases.
Members of the Chinese Academy of Science and the Chinese Academy of Engineering voted the paper the 2nd most important science advance of 2018 (after the Mars landing)
http://news.sciencenet.cn/dz/dznews_photo.aspx?id=31645
Year(s) Of Engagement Activity 2018
URL https://www.imperial.ac.uk/news/186732/new-type-photosynthesis-discovered/
 
Description radio interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact radio interview Radio Sputnik on significance of our paper in Science on far red photosynthesis
Year(s) Of Engagement Activity 2018
 
Description radio interview Australian Broadcasting Company 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact discussed the significance of our publication in Science on long wavelength photosynthesis
Year(s) Of Engagement Activity 2018