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Unformatted text preview: BC368 – Biochemistry of the Cell II Experiment 4 Inhibition of Photosynthetic Electron Transport by Substituted Quinones Introduction Light energy is transformed into chemical energy by the process of photosynthesis. Photosynthesis is initiated by absorption of a photon by one of many possible organic pigments present in the cell. These pigments include the chlorophylls and the carotenoids, the combined absorbances of which cover a large region of the spectrum between 400-700 nm. Light absorbance triggers a series of oxidation-reduction events, resulting ultimately in the oxidation of water to molecular oxygen and hydrogen ions and the concomitant reduction of carbon dioxide. The overall reaction as it occurs in green plants can be represented as follows: CO 2 (g) + H 2 O(l) [CH 2 O](aq) + O 2 (g) Photosynthesis occurs in both green plants and some bacteria. While anaerobic bacteria use substrates other than water as the electron donor, the overall molecular logic is very similar. The mechanism of photosynthesis involves two stages: the “light” reactions, which require light to occur, and the “dark” reactions, which do not require light (but do occur in the daytime!). The light reactions are the focus of this experiment, which will use spinach chloroplasts as the source of the photosynthetic apparatus. Structure of the Chloroplast Spinach chloroplasts are small ellipsoid structures of about 4-10 μ m in diameter and 1 μ m in thickness. A double membrane system encloses the fluid interior, called the stroma, which contains the enzymes of the dark reactions. Isolated chloroplasts contain the internal membranes, called the thylakoid membranes, within which is the machinery of the light reactions. Conveniently, thylakoid membranes can be isolated from green plant material through mechanical fractionation and differential centrifugation. Measurement of Photosynthetic Activity In this exercise, you will be measuring light-induced electron transfer activity in isolated plant chloroplasts by monitoring the reduction of the same artificial electron acceptor that you used in Experiment 3. When chloroplast membranes are isolated, the electron transport chain is left without a terminal electron acceptor. 2,6- dichlorophenolindolphenol (DCPIP) is not only of suitable redox potential to serve this purpose, but its reduction can also be conveniently monitored colorimetrically: E-FADH 2 + DCPIP oxidized E-FAD + DCPIP reduced (blue) (colorless) Cl O Cl N O- Na + oxidized form of DCPIP h υ Δ G = 480 kJ/mol BC 368- Experiment 4- Spring 2010 2 Inhibition of Photosynthesis Electron transport can be inhibited in the presence of certain compounds that act as herbicides. The compounds that you will study in this exercise are structural analogues of the mobile electron carrier Q B , a nonpolar plastoquinone. Q B shuttles reducing equivalents from the membrane-bound protein complex Photosystem II to the cytochrome bf complex. Oxidized Q B binds via specific interactions to one of the protein...
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