Chloropyhll...more like Boraphyll

Well, I am sitting at work right now with tons of down time because we are only prepping samples today, so I figured I would say a little about what I do here on non-down time days.

In this lab, I am working with photosynthesis, and more specifically, Photosystem II (PSII), and more specifically, the Oxygen Evolving Complex (OEC) of PSII, and more specifically, the effect of the Chloride ion (Cl ion) on the OEC. Photosynthesis is, of course, how plants turn solar energy into chemical energy. PSII is the first protein complex in photosynthesis and is where water is oxidized to oxygen, which produces 4 electrons and 4 protons (which are turned into chemical energy). The OEC is the reaction center where water is actually converted to oxygen, and it includes four manganese atoms, one calcium ion, and one chloride ion (the tetramanganese cluster). This cluster is the machinery that turns water into oxygen. The chloride ion has been proven to be necessary in oxygen evolution, though for longer than I have been alive, its actual function has been a mystery.

Here recently published diagram of the conversion of water to oxygen and its release of electrons and protons. This model is called the S-state model (starts with S0).

Most days I do assays on chlorophyll samples, which means testing the oxygen evolution capability of PSII under various conditions. We use a mini-reaction chamber that is hooked up to a Clark electrode, which tests for oxygen. The Clark electrode is interfaced with a graph machine that plots a line based on how much oxygen it senses. From this line, we are able to calculate the rate of oxygen evolution in the reaction chamber. The rate is compared to other rates and is used to determine if the condition tested is suitable.

So what does all that mean? Well, basically, I am testing various protein mutants to try to determine the function of the Cl ion in the tetramanganese cluster in the OEC.

On other days, I actually prepare the samples that we test, which means extracting PSII from spinach leaves. It is a four day process that starts with cleaning and de-stemming spinach leaves and putting them on ice for a day. The next couple days are spent spinning the goo-ified spinach leaves many times at various speeds in the centrifuge. It takes many times, because you have to first separate out the chloroplasts from the cells, then the Reaction Centers (RCs) from the chloroplasts, then PSII from the rest of the RCs, then the starch from everything, then extrinsic proteins from PSII, etc. It is a long process, but actually pretty cool, considering you end up with about 150 mg of one protein complex from a bunch of leaves.

Here is a picture of the washer-sized centrifuges that we use to spin up to 30,000 rpm.

Well, I suppose that is it for now. Sorry if this is not very good explaining, and you are probably more bored than I am right now, but this is really interesting stuff (at least for a nerd).