Photosynthesis in action: new technique resolves atomic changes in undamaged photosystems

All life on Earth begins and ends with photosynthesis, the super-efficient method adopted by plants to convert light energy into its more useful sugar form, with oxygen as the serendipitous byproduct (for us, anyway).  But for so ubiquitous a phenomenon, we understand very little about the microscopic mechanisms that allow this process of light-energy harvesting to be so much more efficient than artificial methods created in laboratories.  The main reason for this lack of understanding is the speed at which these processes take place, too fast for experiments thus far to dissect them at an adequate spatial and temporal resolution.  But help is on the way!  A study in Nature has revealed a new experimental technique, time-resolved crystallography (more on what that is in a moment!), that allow scientists to take snapshots of the innards of photosynthetic systems at the femtosecond time scale.  Let me see if I can guess a few questions you already have…

Figure courtesy of legacy.owensboro.kctcs.edu/

Figure courtesy of legacy.owensboro.kctcs.edu/

How do these photosynthetic systems work?

Converting light energy to more useful forms, either using carbon dioxide to create sugars or through water splitting, seems like a simple process.  But the machinery required in plants is quite complicated.  In fact, we’re still trying to figure out how evolution has crafted such a beautiful machine.  We do know some basics, though.  The central agent involved in this energy conversion is the photosystem.  There are two types – photosystems I and II – that are big protein complexes in the thylakoid membranes in plants and algae (see figure above).  These can be found in plant leaves that capture most of the sun’s rays.  This paper focuses on photosystem II (left side of figure above), which is used to split water into hydrogen and oxygen.  Importantly, these systems contain oxygen-evolving complexes (OEC) that cycle through five different stages of oxidation after photoexcitation, S0 to S4, which together extract and separate four electrons for the ultimate chemical reaction to split water. The OEC is known as a catalyst, meaning its existence dramatically increases the reaction rate.  Catalysts are crucial to any photochemical process, so knowing how the OEC works in plants is of utmost importance.  This study focuses on how the OEC structurally changes throughout the electron excitation and transfer process of the reaction to hopefully better understand how it catalyzes so well.

Figure courtesy of [1]

Figure courtesy of [1]

So why can’t we just replicate this process using other materials?

That’s the million dollar question!  We wish we could, but there’s still a lack of knowledge about what these reaction centers, the photosystems, are actually doing to convert the light energy so quickly and efficiently.  That’s why this new study is ground-breaking – the researchers have developed an experimental technique that allows them to see changes in the shape of the photosystems at the femtosecond to microsecond time scale.  These shape changes relate to different stages of electron transfer – the light gives energy to the photosystem, which excites an electron that can be used for the chemical reaction to create sugars from the carbon dioxide or split water, releasing oxygen in the process as well.

Figure courtesy of www.comicvine.com

Figure courtesy of http://www.comicvine.com

Wait, just how fast is a femtosecond?

A femtosecond is 10^-15 seconds – but that doesn’t help much.  Maybe it’s better to use a comparison.  A femtosecond is to a second what a second is to 31.7 million years!  A second seems fast to us, but imagine all that could be done if we could do meaningful things, like wash the dishes, on the scale of femtoseconds.  The amazing thing is that many chemical processes occur on this time scale, especially electron transfer dynamics, so it’s crucial we develop experimental techniques to see real-time processes at this time scale.

Figure courtesy of [1]

Figure courtesy of [1]

OK, so just what did this experiment do?

Previously, experimentalists have looked at the atomic structure of photosystems using x-ray crystallography.  Basically, this entails shining high-energy light (the x-ray spectrum) at the sample from all different angles and positions.  When the light hits the sample, it diffracts when it hits the sample and is sent in many different directions and angles (the rest is either absorbed or transmitted straight through).  Detectors are set up around the sample that measure how much light is diffracted to their different positions, and this data can be used to reconstruct an image of the sample.  Experiments have used this technique already to image photosystem II up to a resolution of 1.9 Angstroms, which is very good!  The issue is that these x-rays have a lot of energy, and that amount of energy bombarding a material has a good chance to damage said material.  So the information gathered from these experiments could be inaccurate, providing data from a damaged photosystem instead of a healthy working one.

Right, but this you said this experiment used time-resolved crystallography – that’s different?

Exactly – over the past few years, scientists have cleverly found a way around this damaging problem.  They still damage the sample – destroy it actually – but they gain information from the sample prior to destruction!  To do this, researchers use the x-ray free-electron laser (XFEL for short, scientists love acronyms for every new invention).  This is a highly energetic laser beam that shines on the sample of interest, just like crystallography, but done so in extremely short pulses at the femtosecond level.  Millions of these pulses hit the sample, destroying it, but each pulse is so short that it provides data prior to its destruction.  In a sense, we’re gaining information about the state of the sample just before being hit with the light beam.

Great, so researchers can now look at the static structure of molecules at the atomic level.  That’s it?

Not just that.  The beauty of this process is that researchers can then add another level of complexity.  Remember that we’re interested in the electron transfer dynamics of the photosystem – how does its shape change with electronic excitation and charge separation through the system.  To look at temporal dynamics, researchers first shine a lower energy light on the sample to excite an electron, then perform the time-resolved crystallography at varying delay times.  Each delay time will reveal a different shape of the photosystem at various points in this dynamical process.  This then provides the data about conformational changes of the photosystem as the electron dynamics occur.  Cool, huh?

Figure courtesy of [1]

Figure courtesy of [1]

Very cool.  But I want to know how useful it is.  What’d the researchers find out about these photosystems?

The researchers did find some interesting exploratory data, focusing on the Mn4CaO5 cluster that is part of the OEC.  After initial photoexcitation, the researchers found evidence of a shift in the structure from the S1 state (electron density in blue, left figure above) to a possible S3 state (electron density in yellow, right figure) that is still controversial within the research community.  The shift between states seems to involve one ligand moving away from the central Mn cluster (upper yellow arrow in right figure).  This ligand, labeled D170, has been hypothesized to be crucial in the evolution of higher S states.  In addition, the electron density around another ligand, the AB loop, shifts closer to the cluster in the S3 state (the bottom left yellow arrow in the right figure), also indicating this ligand’s importance.

These findings need replication and further study at higher spatial resolutions.  Still, they indicate that this method can be used to gain much more knowledge about that exact shape of these distinct states that are part of the oxygen evolution reaction.  Indeed, the central impact of the paper is more as a proof-of-principle for the new time-resolved technique.  This will open the door to a host of new studies using high-resolution, time-resolved structural changes of photosystems and water oxidation processes.  This is the next step for the mystery of these beautiful and ancient Gaian machines to be understood and appreciated!

P.S. Look at the number of authors on this paper!

References

1)

ResearchBlogging.org

Kupitz, C., Basu, S., Grotjohann, I., Fromme, R., Zatsepin, N., Rendek, K., Hunter, M., Shoeman, R., White, T., Wang, D., James, D., Yang, J., Cobb, D., Reeder, B., Sierra, R., Liu, H., Barty, A., Aquila, A., Deponte, D., Kirian, R., Bari, S., Bergkamp, J., Beyerlein, K., Bogan, M., Caleman, C., Chao, T., Conrad, C., Davis, K., Fleckenstein, H., Galli, L., Hau-Riege, S., Kassemeyer, S., Laksmono, H., Liang, M., Lomb, L., Marchesini, S., Martin, A., Messerschmidt, M., Milathianaki, D., Nass, K., Ros, A., Roy-Chowdhury, S., Schmidt, K., Seibert, M., Steinbrener, J., Stellato, F., Yan, L., Yoon, C., Moore, T., Moore, A., Pushkar, Y., Williams, G., Boutet, S., Doak, R., Weierstall, U., Frank, M., Chapman, H., Spence, J., & Fromme, P. (2014). Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser Nature DOI: 10.1038/nature13453

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