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Ann. Rev. Biophys. Bioeng. 1977. 6:1-6 Copyright © 1977 by Annual Reviews Inc. All righls reserved
DELAYED LIGHT +9084
IN PHOTOSYNTHESIS
William Arnold Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830'
"Delayed light" from green plants was discovered in 1951, by Strehler & Arnold (1), during an attempt to show that illuminated chloroplasts produce ATP. Since the emitted light lasted far too long to come from any excited state of chlorophyll, we concluded that the energy emitted must have been stored in some other form.
The problem of how the light energy absorbed by chlorophyll is stored and then . made available to reduce CO2 to carbohydrate and oxidize water to O2 was already old in 1951. The possibility that the storage of energy in delayed light and in photosynthesis were the same suggested that in delayed light we might have a new probe with which to attack the problem of the first steps in photosynthesis.
For the last 25 years, here in Oak Ridge and in other laboratories all over the world, delayed light has been studied to see what it could tell us about photosynthesis. As might have been expected, delayed light turns out to be complicated. In the book Bioenergetics of Photosynthesis (2), there is a beautiful review by Lavorel of the studies on delayed light, along with a list of approximately 100 references. I recommend this essay to any reader who would like to know more about this light emission.
In what follows, I list some of the principal observations and ideas concerning delayed light and then speculate on the relation between delayed light and photosynthesis.
THE INTENSITY OF DELAYED LIGHT
Green plants that have been illuminated with an exciting intensity in the region where the rate of O2 production is linear with intensity have a quantum yield for fluorescence of about 0.02. Under these conditions the intensity of the delayed light, in the millisecond region, is about 0.01 the intensity of fluorescence. Without the use of photomultiplier tubes, the study of delayed light would be nearly impossible;
with the use of photomultiplier tubes, the measurement of delayed light is one of the simplest observations that can be made ort plants.
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2 ARNOLD
10O,
•
50 •
<J) :!:::: C :J • :>. ... 0 � • -
.0 ... 0 •
� 10 • I <.9 • ..J
•
0 5 w • >- • <t: ..J w 0 •
•
2 3 4 TIME (min) IN DARK
Figure I Delayed light-thl! logarithm of the intensity of the delayed light as a function of the time in the dark. The sample was a 26-mm-Iong cucumber. The curve for a leaf or a chlorella suspension would look much the same.
"He had been eight years upon a project for extracting sun-beams out of cucumbers, which were to be put into vials hermetically sealed, and let out to warm the air in raw inclement summers." From Gulliver's visit to the Grand Academy of Lagado.
THE SPECTRUM OF DELAYED LIGHT
The emission spectrum olf delayed light is the same as the emission spectrum of fluorescence, and the action spectrum is the same as that for oxygen production.
THE DECAY CURVE
The intensity of delayed light is measured as a function of time in the dark, from 10-5 sec to 90 min (3). The curve of log I against time is concave upward over the whole range, i.e. the longer the time in the dark the greater the relaxation time. The curve has some structure consisting of small waves; the intensity of the delayed light changes by something likle 108 over the curve.
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DELAYED LIGHT IN PHOTOSYNTHESIS 3
SATURATION OF DELAYED LIGHT
In photosynthesis, the rate of oxygen production is a linear function of the intensity of the exciting light at low intensities. As the exciting intensity is increased, the rate becomes constant and independent of intensity; this is called saturation. Delayed light saturates in much the same way except that the intensity of exciting light needed to give saturation is a function of the dark time, the time between excitation and measurement. For saturation at 104 sec a l000-W lamp and lens is necessary, whereas for a dark time of 30 min a 5-W lamp at I m will do.
THE MECHANISM OF OELA YEO LIGHT PRODUCTION
From the emission spectrum it is known that the light comes from excited chlorophyll. Hence, the problem is this: How is the stored energy used to make excited chlorophyll? Experiments suggest that there may be four or five different mechanisms involved. I discuss two here: the recombination of electrons and holes, and the untrapping of electrons or holes.
Calvin and Tollin found that plants that were relaxed in the dark for some minutes and then frozen (-100°C) and illuminated with a short bright flash give delayed light (4). The decay of the light is approximately exponential, with a time constant of2 to 3 msec. After repeating the experiment a few times, no more delayed light is emitted unless the plants are warmed and refrozen. To understand this experiment, assume that an excited chlorophyll can give an electron to a trap and leave a free hole, and that an excited chlorophyll can give a hole to a trap and leave a free electron. The free electron and the free hole can recombine to form the excited chlorophyll. The process stops as soon as all of the traps for electrons and holes are filled. Warming the samples allows thermal fluctuations to empty the traps so that the experiment can be repeated. Bertsch, working in Hills laboratory, observed that the delayed light from chloroplasts (measured 1 msec after illumination) is increased several-fold when ferricyanide is added to start the electron flow (5). One would think at first that using stored energy for photosynthesis or the Hill reaction must diminish the intensity of the delayed light; however, if two quanta cooperate then both delayed light and electron flow can increase at the same time.
The untrapping of electrons and holes can be shown by illuminating plants at room temperature and then, while still illuminated, freezing them with liquid nitrogen. The sample then is heated in the dark, at the rate of a few degrees per second, to 100°C. The intensity of emitted light is measured as a function of time. This Glow Curve consists of a number of peaks. Activation energies can be calculated from the rate of heating and the temperature at the time of the maximum for each peak. The activation energies for the four main peaks in the Glow Curve of chlorella are 0.53, 0.60, 0.62, and 0.64 eV. I believe that some of these are the energy needed to lift an electron from the trap back to a block of chlorophyll molecules that contain a free hole, and that some are the energy needed to lift a hole back to a block of chlorophyll containing a free electron.
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4 ARNOLD
THE LIGHT REACTION IN PHOTOSYNTHESIS
In the light reaction, the energy absorbed by chlorophyll is used to oxidize water to O2, to give electrons at -0.4 V, and to produce ATP. There is good evidence that there are two different systems of chlorophyll, known as I and II. In each system the chlorophyll is assembled in small packets called photosynthetic units (PSU). System I PSU seems to have about 1 10 chlorophyll molecules and system II PSU (the system that oxidizes H20) has 400--500 chlorophyll molecules; the linear dimension is about 150 A. In each of the units, the energy of excitation can move from one chlorophyll molecul(� to another so that it can run over the whole unit. Each unit has two reaction centers; one for reduction, where the unit can give up an
.
electron; and one for oxidation, when the unit can give up a hole (take up an electron). Although little is known about the reaction centers in green plants, there is some information about those in photosynthetic bacteria.
I assume that when an excited chlorophyll gives up an electron at a reducing center the positive charge (a hole) left on the chlorophyll molecule can move from
2 . .-------·------------------------------�
Excited State
\
> � >- 1. hll = 1.8 eV (9 a:: w z w
o Ground Slate
Fast
� E= 0.6 eV
Stabilization Energy
1. 2 eV Energy that
can be u sed for
Photosynthesis
Figure 2 Energy levels in chlorophyll and the trap level. The electron dropping from the excited state of chlorophyll t.o the trap gives up about 0.6 eV of energy and stabilizes the system so that the rest of the energy has time to be used in photosynthesis.
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DELAYED LIGHT IN PHOTOSYNTHESIS 5
one chlorophyll to another, just as a hole can move in the valence band of silicon. Also, when an excited chlorophyll gives up a hole (takes up an electron) at an oxidizing center, the extra electron on the chlorophyll molecule can move from one chlorophyll to another just as an electron in the conduction band of a semiconductor. These are called free electrons and free holes. They permit an understanding of the recombination light.
THE RATCHET
The reaction that traps the energy of excited chlorophyll must be very fast in order to compete with fluorescence. It also must be a kind of "ratchet" so that the trapped energy does not flow back to the fluorescent state. The ratchet need not be completely irreversible, as in a clock, but must hold the energy long enough for the next step in photosynthesis to take place. There is an old argument that gives an estimate of the rate. It is known that the quantum yield of fluorescence is about 0.02 when plants are doing photosynthesis at low exciting light intensities. If the fluorescence is measured with very bright, short flashes (10-3 sec) of exciting light, the intensity of fluorescence is found to be four- to fivefold higher. Since the time of the flash (10-3 sec) is much shorter than the turnover time for photosynthesis (20 msec of light saturation), we can assume that only the first quantum absorbed in the flash can lead to photosynthesis; for the rest, it is as if there was no photosynthesis. With no photosynthesis and with 0.1 of the energy going to fluorescence, 0.9 must be going to heat; the heat production is nine times as fast as fluorescence. Therefore, for plants in photosynthesis, fluorescence uses 0.02 and heating uses 0.18; thus photosynthesis uses 0.8. The rate at which energy is trapped for photosynthesis is 40 times faster than the rate of fluorescence and the natural lifetime of chlorophyll calculated from the absorption spectrum is 15.2 X 10-9 sec; therefore, the rate of chlorophyll fluorescence is 6.6 X 107 per sec and the rate of trapping energy for photosynthesis is 2.6 X 109 per sec. This rate is for the PSU.
It is not known how long the trapped electrons and the trapped holes must be stable for photosynthesis; however, from the experiments of Joliot et al (6) and Kok et al (7) on the waves of oxygen production with the flash number, we know that the traps involved in the oxidation of water are stable for longer than 2 sec and are unstable for less than 5 min. Let us take the geometric mean of these two limits (24.5 sec) as the relaxation time for the untrapping of electrons and holes by thermal fluctuations. The rate of un trapping is F e-E1kT
, where F is the frequency factor (108_109 per sec) for electron untrapping, kTis 1/40 of an eVat room temperature, and E is the energy needed to lift an electron from the trap back to the chlorophyll. By making the estimated and calculated relaxation times equal, we have an equation from which we can find E: 24.5 = 10-9 e+40·E or E = 0.60 eV.
The value of E calculated in this way is nicely bracketed by the values of the activation energies from the glow curves. E is the energy used to stabilize the rest of the energy of the absorbed quantum so that there is time for chemical reactions to take place. It would seem that no part of E can be used in photosynthesis without contradicting the second law of thermodynamics.
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6 ARNOLD
CONCLUSION
In the early 1940s, I was privileged to have many discussions with James Franck on the mechanism of photosynthesis. Each time he would ask "What is the ratchet?" He felt that the problem of how green plants stabilize the energy of excited chlorophyll was fundamental to understanding photosynthesis. In this essay, I listed the main phenomenon associated with delayed light and argued that the ratchet is the trapping of electrons and holes, and that delayed light is the untrapping of some fraction of these by thermal fluctuations.
Other authors have made quite different suggestions as to how the excitation energy is stabilized. I feel that those theories in which the energy of two quanta are combined (by triplet-triplet or singlet-triplet interaction) before the act of stabilization are unlikely, since photosynthesis can take place at very low intensities of exciting light. However, since the astronomers have found synchroton radiation and stimulated emission occurring in various parts of the galaxy, and since there has been an extensive R&D program to increase the efficiency of photosynthesis carried out by the green plants thl�mselves over the last few billion years, it may be that not all the physics needed to understand photosynthesis is in the elementary texts.
ACKNOWLEDGMENT
This research was sponsored by the United States Energy Research and Development Administration under contract with the Union Carbide Corporation.
Literature Cited
I. Strehler, B., Arnold, W. 1951. J. Gen. Physiol. 34:809, 820
2. Govindjee. 1975. Bioenergetics of Photosynthesis. New York: Academic. pp. 223,317
3. Arnold, W., Davidsoll1, J. B. 1963. Photosynthesis Mechanisms in Green Plants, publication 11145. National Academy of Sciences, National Research Council 697-700
4. Tollin, G. Personal communication 5. Bertsch, W., West, J., Hill, R. 1969.
Biochim. Biophys. Acta 172:525, 538 6. Joliot, P., Joliot, A., Bouges, B., Bar
bieri. G. 1971. Photochem. Photobiol. 14:287, 305
7. Kok, B., Forbush, B., McGloin. M. 1971. Photochem. Photobiol. 14:307, 321
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