Photosynthetic water oxidation driven by near infra-red light

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.

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.

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.