Let’s start by accessing your long-term memory, dragging out some of that basic biology information you buried after high school and grabbing on to that dusty file about photosynthesis. If you remember, plants have little green, bean-shaped energy factories in their cells called chloroplasts. These chloroplasts are filled thylakoids stacked up in grana. The thylakoid membranes contain networks of pigments, including chlorophyll, arranged in aggregates or complexes. Think of them kinda like light energy harvesters. Energy is captured for functional and structural units of protein complexes called Photosystem I (PSI) and Photosystem II (PSII). PSI is the light reaction and converts light energy to chemical energy. The pigments of the complexes each absorb light and then pass along that light energy to the central chlorophyll molecule to do photosynthesis. The energy obtained in this reaction is stored in ATP (adenosine triphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate-oxidase) molecules. PSII, the dark reaction, takes place in the stroma within the chloroplast. This reaction uses the Calvin cycle to convert carbon dioxide and energy from ATP into glucose (sugar). To say that is photosynthesis put shortly and simply would be an understatement, but keep this basic reaction in mind:
6 CO2 + 6 H2O à C6H12O6 + 6 O2
It is important to mention that in PSII, water is photochemically oxidized to dioxygen (O2) by the oxygen-evolving complex (OEC), a metalloenzyme cluster containing manganese and calcium. The OEC cycles through five photo-catalytic stages (S0-S4) in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events by a repeatedly photo-oxidized chlorophyll center (Kok cycle). This is the reaction that makes all that oxygen we breathe.
2 H2O à S0-S4 à O2 + 4 H+ + 4 e-
|Photo by: Mary Zhu @ ASU|
The new paper by Kupitz et al. (and al. and al. and al.) published in Nature looks closer (very close!) at this PSII water-splitting reaction. They had some issues to overcome if they wanted to collect more information on this reaction, mostly involving the static nature of X-ray crystallography and the damage done to the OEC with this method. Traditional X-ray crystallography enables 1.9Å resolution (near atomic) but the OEC probably suffers X-ray damage. To overcome this, the researchers used serial femtosecond crystallography. This method uses single shot diffraction patterns are collected from a stream of nanocrystals, using 120 Hz femtosecond (one millionth of a nanosecond!!) pulses from an X-ray Free Electron Laser (XFEL). The second is the quality of the structural information. These pulses are so intense that the sample/specimen is destroyed, but the pulse duration is so short that the diffraction is observed before the destruction occurs. The method produces millions of “snapshots” in hours and can collect time-resolved data for dynamic processes like water oxidation in PSII.
The researchers developed a multiple-laser illumination scheme to observe this dynamic reaction in thermophilic cyanobacterium (Thermosynechococcus elongates). They progressively excited the OEC in dark-adapted PSII nano/microcrystals by two laser pulses from the dark S1 state via the S2 state to the double-flash putative S3 state (5 and 5.5Å resolution). Believe it or not, that was their method put simply. Essentially, they were able to determine the structures of the states and to produce maps of the protein subunits and cofactors of PSII, including the electron transport chain. They found that PSII undergoes significant conformational changes electron acceptor side and at the Mn4CaO5 core of the OEC. The metal cluster significantly elongates, making room and allowing for binding of the incoming water molecules. Then voilà! Water splitting!
So I know what you may be thinking: Why all of that lead-up to a simple protein shape change conclusion? Well, it’s all about mechanism, figuring out the process of photosynthesis at its most basic level. If you think about it, photosynthesis is the biological reaction. It is fundamental to life on Earth as we know it. It converted the oxygen-poor atmosphere of early Earth to the oxygen-rich atmosphere we (and all other respiring organisms) depend on, and continues to supply us with life-giving oxygen. That oxygen comes from this water splitting reaction, and the OEC is one of those structures where you usually have to but "possible model of..." in front. This type of study gives incredible resolution of this structure as well as a new methodology to gain further knowledge. With a more technological viewpoint, work like this could eventually lead to the development of an artificial leaf and synthetic photosynthesis. And, let’s face it, that is really really cool.
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
Arizona State University Science and Tech press release: "ASU-led study yields first snapshots of water splitting in photosynthesis"
Science Daily article: “First snapshots of water splitting in photosynthesis”
For more on the plant physiology:
ASU's page "What is Photosynthesis?"
James Johnson @ FSU page on "The Manganese-calcium oxide cluster of Photosystem II"
Dr. Jakubowski at Saint Johns University "Chapter 8 - Oxidation/Phosphorylation"
(images via FSU Molecular Expressions, Jakubowski's website, and Arizona State University, respectively)