Capturing the Soul of Photosystem II

Something new under the sun… techniques for ‘photographing’ the PSII water splitting reaction.

Some cultures believe that taking your photograph captures part of your soul, and believers shun having their images captured to avoid any potential metaphysical rifts. If this is the case, I suppose it’s safe to say that the world is quite soulless based on the number of selfies floating around the internet these days. As it turns out scientists in my field are having a similar problem with a longstanding question regarding my favorite enzyme, Photosystem II (PSII). We need a technique for photographing PSII that doesn’t destroy its soul (catalytic center- that is).

Note, this is not the actual set up devised by Kern and colleagues

Note, this is not the actual set up devised by Kern and colleagues

PSII is a multi-subunit membrane protein complex that uses light energy to split water into protons (H+) and molecular oxygen (O2). This important reaction makes it possible for aerobic life on earth.* In order to drive this reaction, a huge redox potential is required and P680, the reaction center of PSII, is the most powerful biological oxidant (1.3 V) identified to date. Mechanistically, this is an extremely difficult reaction to do. Using four successive photons, PSII must sequentially pull four electrons from two water molecules and form an O-O bond. All of this is accomplished by the Oxygen-Evolving Complex within PSII, an inorganic cluster of four manganese atoms and one calcium atom (Mn4CaO5). This works because manganese is a transitional metal that can exist in a number of different oxidation states Mn2+, Mn3+ and Mn4+. This kind of stable redox space makes it the perfect element for holding onto the four electrons required to split water and form O2. Unraveling the mechanism by which PSII does this is one of the holy grails in our field. If we only knew how it worked, we could make artificial systems that take advantage of the mechanism for coupling light energy to electron extraction from water (translation = ‘free’ electrical energy from water).

Because of the potential applications, many scientists have been working for many years to figure out how this works. We know some things about the mechanism, but the details we need to know rely on having high resolution structural data on PSII as it is working. We’re not there yet. The currently available experimental tools fall into one of two broad categories. The first is X-ray diffraction structure data or protein crystal structures that represent a static picture of the entire complex (proteins and cofactors). The second is X-ray spectroscopy (absorption or emission). These experiments provide detailed information on the Mn4CaO5 cluster, but not really a picture- just distance constraints between atoms.

Over the past decade, photosynthesis researchers have made great strides in improving the pictures we have using these techniques, but there are some significant limitations in the data. The problem is twofold. The X-rays used to generate our beautiful high resolution (1.9 Å) crystal structure model destroys the Mn4CaO5 cluster during the process of collecting data. We know from other techniques that the energy in those X-rays reduces the Mn4CaO5 cluster to a combination of oxidation states that are not useful for water splitting. On the other hand, the other X-ray spectroscopy techniques are not damaging to the Mn4CaO5 cluster, but it doesn’t give a singular picture and multiple models can fit into the distance constraints provided by those experiments. Thus, the pictures that we get from these experiments are inaccurate or inadequate to answer the question of how PSII splits water. Plus, we would like to visually capture the process as the enzyme turns over at physiological conditions (as in not cryogenic temperatures).

Today’s post highlight’s a presentation from the Midwest Photosynthesis Meeting presented by plenary speaker Jan Kern from Lawrence Berkley National Laboratory (LBNL). He along with numerous other teams of researchers have been working to develop the techniques necessary to take snapshots of PSII as it is splitting water in a way that doesn’t destroy the enzyme. Their system will simultaneously collect data using the two different techniques (X-ray diffraction and X-ray Emission) on the same sample in such a way that the PSII Mn4CaO5 cluster isn’t destroyed. The secret to this is the use of ultra-short high-intensity X-ray pulses. It’s like having a camera that captures the image so fast you don’t even have time to blink before the flash. Also, the system is designed such that the sample can be illuminated with light for varying numbers of flashes to capture images of PSII during different steps of its catalytic cycle. This has been an enormous effort to design the proper experimental set-up and get it working. The preliminary data and proof of concept were published earlier this year in Science. Don’t get ready to start making electricity from water just yet. The system has not achieved the necessary resolution to see the fine structural changes of the Mn4CaO5 cluster as it splits water. However, Jan Kern and others at LBNL are working hard to tweak the components of their system so it does have the necessary resolution for the snapshots we need. Check out the links below for more details on how they did it.


*That’s you and me!


Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode


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