In the Laboratory
Measurement of Quantum Yield, Quantum Requirement, and Energetic Efficiency of the O2-Evolving System of Photosynthesis by a Simple Dye Reaction A. Ros Barceló* and J. M. Zapata Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain Photosynthesis is the conversion of absorbed radiant energy from sunlight into various forms of chemical energy by the chloroplasts of higher green plants. The overall process of photosynthesis consists of the oxidation of water (with the release of O2 as a product) and the reduction of CO2 to form organic compounds such as carbohydrates (1). Thus, photosynthesis uses light energy to drive electrons uphill from H2O (E09 = +0.816 V) to a weaker electron acceptor CO2. Although in the intact leaf the reactions that make up O2 evolution are closely coupled to the reactions comprising CO2 reduction, in the test tube electrons produced by the photolytic cleavage of H2O may be diverted from their true acceptor by inserting a suitable dye in the electron chain. A suitable acceptor that acts as electron scavenger is the dye 2,6-dichlorophenol indophenol (DCPIP) (E 09 = +0.217 V) (2), which is blue in the oxidized quinone form (I, Fig. 1) and becomes colorless when reduced to the phenolic form (II, Fig. 1). This dye electron-acceptor also has the advantage that it accepts electrons directly from the quinone (Qa) electron-acceptor of the photosystem II (Fig. 2), the reaction center associated with the O2-evolving (or water-splitting) system (3). Thus, in the presence of DCPIP, O 2-evolution during photosynthesis may be written as DCPIP (blue) + H 2O → DCPIP{H2(colorless) + 1⁄2 O 2 (1) Since this reaction is endoergonic (∆G° = 231 kJ mol{1 O2 and therefore ∆G° > 0), the reaction requires a supply of energy (light) and the proper catalysts (the chloroplast). Bearing eq 1 in mind, the quantum yield (Φ) in O2-evolution may be easily calculated from
µmol O 2 s –1 (2) µmol photons s –1 the µmol of O2 produced being calculated from the stoichiometry of DCPIP bleaching. Likewise, the energetic efficiency (f) Φ=
f =
chemical energy stored light energy supplied
(3)
may also be calculated. Procedure Estimation of DCPIP bleaching is carried out colorimetrically at 600 nm (ε600 = 22,000 M{1 cm{1 for DCPIP) (2). For this, a petri dish ([ = 9.0 cm) containing 15.0 mL of 0.1 mM DCPIP in 50 mM Na-phosphate buffer, pH 6.50, and 5.0 mL of chloroplast suspension containing active photosystem II, is placed in a water bath at 25 °C. The dish is illuminated with a radiant flux (radiant energy received) of 2.0 J s {1 of red light for exactly 10 min, by interposing a CBS red 650-nm filter (Ref. 68-6710; Carolina Biological Supply Company, *Corresponding author.
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Figure 1. Oxidoreduction pair of 2,6-dichlorophenol indophenol showing its oxidized blue quinone form (I) and its reduced colorless phenolic form (II).
Figure 2. Organization of the electron transport chain in the thylakoid membrane (t) of the chloroplast, showing the normal electron flow (dotted arrow) from the O2-evolving system (1) to ferredoxin (7). In this chain, the dye 2,6-dichlorophenol indophenol (Dox ) acts as electron scavenger at the level of the pool of quinones (3), which receive electrons from photosystem II (2) and which, in the absence of the dye, are saved by the quinone pool and are transferred consecutively to the cytochrome bf complex (4), to the plastocyanin pool (5), and finally to photosystem I (6). Electron transport through photosystems I and II occurs only in the presence of photosynthetically active radiation (hv). In this drawing, the asymmetry of the thylakoid membrane is illustrated by means of the reactions that occur in the intrathylakoidal (i) and stroma (s) face.
Gladstone, OR) between a 100-W cool-white light and the petri dish (Fig. 3). Radiant flux on the petri dish is easily measured with a commercial radiometer and may be varied by moving the petri dish closer to or farther from the illumination source. After illumination, the absorbance at 600 nm is read. As a control, the same procedure—except for illumination—is carried out in the dark. For this assay, chloroplasts containing active photosystem II are prepared following a published laboratory protocol (4). Remove the large veins from spinach leaves. Homogenize 5 g of leaves by grinding in a mortar with a pestle, in an ice bath, in 50 mL of 50 mM Na-
Journal of Chemical Education • Vol. 73 No. 11 November 1996
In the Laboratory
ing that for each mole of 650-nm photons supplied, only 0.097 × 10{3 moles of O2 is evolved during photosynthesis. In other words, the quantum requirement (1/Φ ≅ 10,309) suggests that 10,309 mol of 650-nm photons is required for the evolution of 1 mol of O2 from H2O. This value contrasts with the theoretical (ideal) quantum requirement, which suggests that 4 mol of red photons is necessary to split 2 mol of water. It is necessary to point out that 1 quantum of red light is the minimum necessary to remove 1 electron from water (3). Finally, the energetic efficiency (f) of the O2-evolving system may also be easily calculated from eq 3, where the chemical energy stored would be given by ∆G° = 231 kJ mol{1 O2 for the reaction described in eq 1, and the light energy supplied by E650 (= 2 × 10{3 kJ s{1). Thus
f =
231 kJ mol –1 O 2 × 1.053 × 10 –9 mol O 2 s –1 = 2 × 10 –3 kJ s –1
0.121 × 10 –3 (J chemical energy / J light energy) Fig. 3. Laboratory equipment for measurement of the quantum yield of photosynthesis: the illumination source (1), the red filter (2), the petri dish containing the components of the reaction (3), and the thermostatized water bath (5) containing the supporting grille (4).
phosphate buffer, pH 6.50. Filter the homogenate through 2 layers of cheesecloth and collect the filtrate in centrifuge bottles or tubes. Centrifuge the filtrate in the cold at 2,000 × g for 5 min. Discard the supernatant and gently suspend the chloroplast pellet in 50 mL of cold phosphate buffer. Repeat the centrifugation and resuspension steps. Prepare the final chloroplast suspension at a concentration of 50 µg chlorophyll mL{1 using the approximate equation 1 µg chlorophyll mL{1 = 28.98 A652 where A652 is the absorbance of the chloroplast suspension at 652 nm. Keep the chloroplast suspension on ice until needed. Results and Discussion After illumination with red light for 10 min, the absorbance at 600 nm due to DCPIP (total absorbance of the reaction medium minus absorbance due to chloroplast suspension) decays from about 1.600 ± 0.100 to 0.210 ± 0.050. This decay in absorbance is due to a lightinduced reduction of DCPIP, since no changes in absorbance are found in controls in the dark. From this absorbance decay and using the Lambert–Beer law (A600 = ε600 [DCPIP] for a cuvette path length of 1 cm), changes in DCPIP concentration, and therefore the rate of DCPIP reduction, can be calculated. This yields a rate of 2.106 × 10{3 µmol s {1 of DCPIP reduced, and from the stoichiometry of this reduction (eq 1), a rate of 1.053 × 10{3 µmol s {1 for O2 evolution is calculated. Likewise, and using Planck’s equation
µmol photons s –1 =
λE 650 × 10 6 N hc
(4)
the rate of supply of photons of 650 nm is calculated to be 10.870 µmol s{1, where λ = 650 nm, E650 = 2 × 10{3 kJ s {1, and Nhc = 119,600 kJ mol{1 nm. With these two values, and from eq 2, the quantum yield (Φ) in O2 evolution during photosynthesis is calculated to be 0.097 × 10{3 µmol O2/µmol photons, indicat-
which also contrasts with the theoretical (ideal) energetic efficacy
f = ∆G° = 231 kJ mol –1 = 0.314 4 Γ 650 4 × 184 kJ mol where Γ is the energy of 1 mol of photons of 650 nm, calculated from eq 4. Although values for the quantum yield, the quantum requirement, and the energetic efficiency calculated in the classroom laboratory differ widely from those expected theoretically—mainly owing to the high radiant flux necessary to allow the laboratory experiment to be completed in a few minutes, which inevitably produces an important loss of energy as chlorophyll fluorescence— these calculations are useful for illustrating the transformation of light energy into chemical energy by the chloroplasts of green plants. (Also contributing to the lower yields are the absorption of red light by the dye DCPIP (λmax = 600 nm) (2) and the scattering of the radiant energy caused by the particulate nature of the suspension of chloroplasts.) A more sophisticated measurement of the quantum efficiency of photosynthesis in plants has become possible through fluorescence analysis of photosystem II (5). Fluorescence probes for measuring quantum efficiencies are based on the fact that energy absorbed by chlorophyll, except under very low light intensity, exceeds that which can be used photochemically. The excess energy is dissipated as heat, thus avoiding damage to photosystem II. Therefore the fluorescence signal, properly analyzed, is a sensitive indicator of the amount of energy being used photochemically and the amount being dissipated (5). Although the theoretical maximum quantum yield for photosystem II is 1 electron per photon absorbed by photosystem II, in practice, the maximum values obtained experimentally from fluorescence analysis for the in vivo quantum yield of photosystem II are around 0.85 (5). However, the laboratory equipment necessary for these experiments and further calculations is far more sophisticated than that usually found in the undergraduate classrooms. –1
Literature Cited 1. Arnon, D. I. Sci. Am. 1960, 203(5), 104. 2. Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. Data for Biochemical Research; Oxford Science: Oxford, 1993; p 352. 3. Rutherford, A. W. TIBS 1989, 14, 227. 4. Moore, T. C. Research Experiences in Plant Physiology; Springer: New York, 1981; p 325. 5. Krall, J. P.; Edwards, G. E. Physiol. Plant. 1992, 86, 180.
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