Secrets of photosynthesis light the way
Decade-long investment from BBSRC opens up new frontiers in ultrafast protein dynamic imaging.
“Let there be light!” This phrase, whether you’re religiously inclined or not, has equivalents in many languages and is the motto of more than 40 educational institutions across the world. It can be seen as recognition that light is the powerhouse that makes all life on earth possible via photosynthesis, the process by which plants make energy from light photons.
However, like many products of natural evolution, photosynthesis is a miraculously efficient but not perfectly efficient process. There are slightly different forms of it in different plant groups across the world: the cluster of photosynthetic enzymes from the wheat in your daily bread is different from that in maize, another of the world’s most important food crops.
For decades, scientists have taken a keen interest in elucidating exactly what is happening in these photosynthetic enzymatic complexes at the molecular level. Now, new technology and techniques are yielding results that could inform work by other researchers in everything from engineering hybrid and artificial photosynthesis to manufacturing even more efficient solar cells.
“Natural photosynthesis is extremely complicated and many individual pigments are involved to collect and eventually chemically store the energy of single photons,” says Dr Jasper van Thor based at Imperial College London. “In order to bring experiment and theory closer together, we developed the ability to find real-space, structural information of the flow of energy in photosynthesis.”
Using BBSRC funding, van Thor and colleagues have developed a unique imaging system that is unrivalled anywhere in the world, built and developed over more than seven years, together with necessary new theory, which can see photosynthetic machinery embedded in crystals for the first time. This allows imaging at the femtosecond level: that’s with a time resolution of femtoseconds - a staggering one quadrillionth, or one millionth of one billionth, of a second!
The researchers wanted to answer fundamental questions about photosynthesis: where does the ‘bottleneck’ for the process occur? After that, what is it about these structures that makes this happen? How can this knowledge best be utilised for future material science and green energy research?
“Decades of photosynthesis research have divided the field, resulting in two opposing possibilities,” says van Thor.
The debate has revolved around whether processes in parts of enzymes called reaction centres contain the bottleneck, or whether the energy transfer to the reaction centres is the bottleneck. It’s a bit like a supermarket trip: is it shuffling down the queue or the actual transaction time at the till that’s slowing up your total shopping time? (See box ‘Photonic cradle’ for more details.)
“We discovered that, with ultrafast infrared measurements of single crystals of Photosystem II core complexes [where the key photosynthetic reactions take place], we could retrieve real-space information on femto- and picosecond timescales of the energy transfer process,” says van Thor. “The overall bottleneck is the energy transfer step to the reaction centre.”
This, adds van Thor, confirms advanced structure-based theory previously developed by one of his collaborators and Nature Communications paper co-author Thomas Renger from JKU-Linz University.
Dividing the light from the darkness
Researchers across the world have been trying to answer this question for decades. So how did van Thor and colleagues manage it? It took time, patience and investment from BBSRC and other funders, namely the Leverhulme Trust. Altogether, the work took more than seven years from conception, proposal, research and theory through to publication and press release.
To realise real-time dynamic images of energy transfer in photosynthesis, van Thor and collaborators brought together three areas of research. “By marrying femtosecond spectroscopy, X-ray crystallography and chemical physics theory, we showed that it was possible to orient micrometre-sized crystals with known X-ray crystallographic index,” he explains. “Then we used this to make sensitive time-resolved infrared measurements of tiny crystals, applying optical crystallography analysis in the X-ray crystallographic frame. Finally, we extended the structure-based theory of light harvesting to apply it to single crystal measurement."
The result can be described as a ‘movie’ of energy transfer, running over a full period of a nanosecond [one thousand-millionth of a second], with femtosecond time resolution.
“BBSRC supported my proposal and vision,” says van Thor. “I developed the scientific case to construct a world-unique instrument specifically for the interesting problem of photosynthesis. Because I was proposing to construct an instrument that did not yet exist elsewhere, proof of principle needed to be shown. The resulting instrumentation constructed was world-unique for several characteristics.”
The resulting instrument is a world-first: a collection of custom lasers, systems and non-linear crystals that has the ability to provide a very brief visible light pulse, followed by a very brief mid-infrared pulse, which can obtain a measurement in a very small spot size. “All together, we can make very sensitive femtosecond infrared structural measurements of micrometre-sized protein crystals,” says van Thor.
Tripping the light fantastic
Now that a new frontier has been reached and breached with van Thor’s BBSRC-funded work, the ultimate goals of understanding the underlying physics of light harvesting can be utilised to design better artificial and hybrid photosynthesis and also solar cell technology, which is an area other BBSRC funded researchers are working on.
And it’s not just limited to the proteins involved in photosynthesis. The technique and equipment can be applied to other proteins and enzymes. “Having subsequently developed the necessary theory and analysis, we now have the special capability at Imperial College London to execute and understand ultrafast infrared-optical crystallography as a structurally sensitive method to probe protein dynamics,” says van Thor.
This means it could be used for imaging and understanding cell receptors that control what drugs and molecules enter and exit cells, or in drug design to see how well compounds dock with targets, or how energy is formed in mitochondria within cells – the applications are many in the world of bioscience, because proteins are where the action is. Fortunately, van Thor’s lab has a new BBSRC grant to further explore and develop this fledgling ultrafast imaging field.
“We are also performing femtosecond time resolved X-ray crystallography which can work for reactive systems,” says van Thor. “In the end we want to know how proteins work, and for technical reasons the crystalline environment must be chosen to answer the questions that we are asking, whether we use infrared, visible, or X-ray methods to visualise ultrafast dynamics in time and space.”
A vital part of photosynthesis is using light energy to split water into oxygen and hydrogen, which is undertaken by a complex called Photosystem II. Light energy is harvested by chlorophyll pigments acting as ‘antennae’, and transferred to the reaction centre of Photosystem II, which strips electrons from water. The first step of this conversion of excitation energy into chemical energy is known as ‘charge separation’.
It was previously thought that the process of charge separation in the reaction centre was a ‘bottleneck’ in photosynthesis - the slowest step in the process - rather than the transfer of energy along the antennae to the reaction centre.
Using ultrafast imaging, scientists from Imperial college London and Johannes Kepler University (JKU), Austria, have shown that the slowest step is in fact when plants harvest light and transfer its energy through the antennae to the reaction centre, not processes in the reaction centre itself. This finding has replaced what used to be a textbook description of photosynthesis.
These images are protected by copyright law and may be used with acknowledgement.
The ultra-fast laser imaging equipment at the Imperial College London
Tags: frontier bioscience Imperial College London molecular bioscience plants image gallery press release