Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4502-4505, December 1976 Botany Path of electrons in (energy levels of /delayed light/semiconductors/carotene diode/system I and II) WILLIAM ARNOLD Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 Contributed by William Arnold, September 20, 1976

ABSTRACT Electrons, from the oxidation of , inside The redox level for the oxidation of water to 02 is at +0.815 the grana disks () are transferred across the membrane V. It is generally found that to make the reaction go, an over- to the outside, to the or the Hill oxidant. The span voltage of about 0.5 V is needed. in redox level may be 2.3 V. Part of the system II chlorophyll is on the inside of the membrane and part on the outside. An The chlorophyll apparatus must be able to lift an electron electron trap is embedded in the membrane. Alternately, an from +1.3 to -1.0 V, a total span of about 2.3 V. excited chlorophyll on the inside gives an electron to the trap, Enhancement. Emerson discovered that with monochro- and an excited chlorophyll on the outside gives a hole to the trap. as one goes through the spectrum the 02 production Two quanta move an electron from inside to outside. The matic light, charging of this condenser drives the redox levels on the inside goes to zero at a shorter wavelength than the absorption of positive and those on the outside negative. The final voltages chlorophyll does (3). That is, some of the longer wavelength depend upon the electron flow and a carotene diode. A voltage light absorbed by chlorophyll cannot give 02- Nevertheless, the of 0.3 is involved. Delayed light is the exact reverse of the light mixing of this non-02-producing light with light that can pro- reaction. System I makes ATP. duce 02 gives a higher quantum yield than either light alone. Photosynthesis is the process by which green plants reduce CO2 This unexpected and baffling phenomenon, now known as to carbohydrates and oxidize water to 02. This process makes "enhancement," has been the subject of much research since the food we eat and the 02 we breathe, and in past times made its discovery. It is believed that enhancement shows that there the coal and oil we now use. Energy, which comes from sunlight are two photochemical reactions in green plants (4). The absorbed by chlorophyll, is stored to the extent of some 5.1 eV long-wavelength (6850 A; 1.81 eV) reaction, system I, does not per carbon atom. produce 02, and from the measurement of Weaver has about Research with radioactive carbon has made it possible to 110 chlorophyll molecules in the unit (5). System II, the short- follow "The Path of Carbon in Photosynthesis" in detail (1). wavelength (6700 A; 1.85 eV) reaction, does produce 02, and CO2 does not take part in a photochemical reaction. Carbon from general arguments about photosynthetic units must have reduction is a series of reactions known as the Calvin about 500 chlorophyll molecules. cycle. This cycle can take place in the dark, and is driven by In a number of the attempts to explain the operation of the electrons at -0.4 V and by ATP. The chlorophyll apparatus (in chlorophyll apparatus, the assumption has been that system II the ) must be able to use light energy to oxidize and system I are connected in series for the electron flow. Sys- water to 02, lift electrons from the level of water (+0.8 V) to tem II has 4.5 times as many chlorophyll molecules as system -0.4 V, and make ATP. I. So an additional assumption has to be made-that system II Exactly how the chlorophyll apparatus works is not known. can transfer excitation to system I-for the rate of electron Many attempts to explain its operation have been published, transfer to be equal in the two systems. The energy difference but none, including two that I have written, are satisfactory. (0.04 V) between the two systems is not large enough to prevent The present paper gives a new map for the flow of electrons the transfer of excitation energy from system I to system II. So in the chlorophyll apparatus. The paper is not a complete theory we should expect to see 02 production [exp(-0.04/kT) = 0.2] of photosynthesis, but an attempt to explain how the energy of at wavelengths where only system I can absorb light. This we light, absorbed by chlorophyll, is made available for chemical do not see. reaction. In this paper, it is assumed that system II and system I are not There are four major constraints that we use in drawing the in series. map. Energy Levels of Chlorophyll. It has been known since the Delayed Light. After illumination, green plants emit, in the time of Stokes that the chlorophyll in green plants fluoresces. dark, a dim glow that we call delayed light (6). The emission It is known that the intensity of the fluorescence depends on the spectrum of this light is the same as that for the fluorescence rate of photosynthesis. We assume that the excitation of chlo- of chlorophyll in the plants (7). This means that excited chlo- rophyll to the singlet state (1.8 eV) is the first step in photo- rophyll must be regenerated in the dark. After bright illumi- synthesis. nation, the intensity of delayed light is proportional to the re- The Hill Reaction: The Span in Redox Potential. Hill dis- ciprocal of the time in the dark, over the range of a few milli- covered that chloroplasts could be removed from plant cells and seconds to 1 1k hr. made to produce 02 in the light if they were provided with The intensity of the delayed light, at time t in the dark, be- some reducible substance (2). These Hill oxidants seem to take comes saturated with respect to the intensity of the exciting the place of the entire Calvin cycle. Many Hill oxidants are now light. That is, we can find an exciting intensity beyond which in redox some are much there is no increase in the delayed light. If t is small, the in- known, covering a wide range levels; If t is more negative than the -0.4 V of the Calvin cycle, extending tensity of exciting light to give saturation is very high. V. large, the intensity of exciting light to give saturation is very to about -1.0 low. Abbreviation: PSU, photosynthetic unit. Experiments show that most, if not all, of the delayed light 4502 Downloaded by guest on October 1, 2021 Botany: Arnold Proc. Natl. Acad. Sci. USA 73 (1976) 4503

Insideizing Outside ch-- ch Oxidizing~_ LI]Reducing -1.0-1 ch*f- chi

-5 -0.5-

- eV 1. 8eV 150 A JK TRAP Q x 0 0 -____ uJ ~~Protein r +05- (nsulator) ctf-- ch*

ch - ch + 1.0- Chlorophyll Lipid Membrane- (250 molecules) (insulator)

FIG. 1. Left side shows the four levels of chlorophyll on the redox scale. Right side is a schematic of a possible cross section of system II PSU.

is made by system II. Experiments also show that the delayed After the reaction the reducing level ch--ch is left. The light is much the same in chloroplasts in which the electron flow transfer of electrons from this level is labeled as slow. has been stopped by poisons. Fig. 1 (left) is a plot of the four levels on the redox scale. The fundamental assumption of this paper is that delayed I light is made by the reverse of the fast reaction that stabilizes System the energy of excited chlorophyll so that it can be used for It is generally believed that the illumination of system I reduces photosynthesis. (-0.4 V) and oxidizes cytochromef (+0.4 V). A fast Energy levels of chlorophyll transfer of an electron from the ch*-ch+ level (-0.85 V) to ferredoxin, followed by a slow transfer of a hole from the To draw the map of the flow of electrons in the chlorophyll ch-ch+ level (+0.95 V) to f, will do nicely. apparatus, I must plot the various chlorophyll levels on the redox scale. Nelson measured the ionization energy and the electron System 1I affinity of solid ethyl.chlorophyllide (8). In a later paper, Azzi I think that the system II photosynthetic units (PSUs) are located and I used his data to estimate where the chlorophyll levels are on or in the membranes of the grana disks. Each disk is a small on the redox scale (9). These values will be used here. vesicle () shaped like a coin some 0.5 AsM in diameter. Nelson emphasized that if excited chlorophyll is the first actor If we take the diameter of one PSU to be 150 A, then there will in photosynthesis and if only one electron at a time is trans- be room for about 800 PSUs on each face of the disk. I think that ferred, then there are four different redox levels. the oxidation of water to 02 takes place inside of the vesicle. The The four levels will be denoted with symbols like "ch--ch," system II PSUs transfer electrons across the membrane, from which means that a chlorophyll molecule with an extra electron inside to outside, when the electrons are used by the Calvin can give the electron to some substance and reduce it, thereby cycle or the Hill reaction. Fig. 1 (right) is a possible cross section becoming a neutral chlorophyll molecule. The left term in the of a system II unit. symbol shows the chlorophyll when the level is filled, the right I assume that some of the 500 chlorophyll molecules are on when the level is empty. one side of the membrane and some are on the other. Light The symbol ch-ch+ denotes an oxidizing level. A chlorophyll energy absorbed by one chlorophyll can travel by resonance molecule that has a positive charge (that is short one electron) transfer to any other. This means that energy can flow freely can become neutral chlorophyll by giving up a hole (taking an across the membrane. Furthermore, some fractions of chloro- electron) and thus oxidizing something. phyll molecules on each side can act as semiconductors, and Excited chlorophyll can act as a reducing level, ch*-ch+ (ch* transfer electrons or holes from one molecule to the next. I also = excited chlorophyll), by giving up the electron that has been assume that the membrane is a good electrical insulator, as is promoted. The electron transfer must be very fast so as to the protein between the chlorophyll and the water on each compete with fluorescence. side. A simple calculation shows that the electron transfer must The square JK is the mechanism that oxidizes water, that be to a level 0.5-0.6 V more positive to allow enough time for Joliot and Kok are now studying (10, 11). Q is the substance that chemical reaction. On the map this electron transfer is labeled takes electrons from system II. T is an electron trap that is in as fast. After the fast transfer from the ch*-ch+ level, the oxi- the membrane. dizing level ch-ch+ is left. Excited chlorophyll can also act as an oxidizing level ch-- Operation of system 11-the map ch*. Crudely, we can think of the neutral excited molecule as Assume that in completely relaxed plants the four redox levels having an electron in a high energy state and a hole in the for the chlorophyll inside and outside are as shown in Fig. 1 ground state. The excited chlorophyll can take an electron (give (left), and the redox level of the trap is in the middle at -0.05 up a hole) to fill the ground state and leave ch-. Again this must V. be a very fast reaction with an energy difference of about 0.5 Assume that when the system is illuminated, the fast reaction V. This reaction is labeled as fast on the map. transfers electrons from the ch*-ch+ level of the inside chlo- Downloaded by guest on October 1, 2021 4504 Botany: Arnold Proc. Natl. Acad. Sci. USA 73 (1976)

-0.5- ChnCyl

x P ~~~~~~~~~~~~~~~~~~~~~or 0 Hill Oxidant 0 +5H20 S/ch--0 -ch+ i~~~~~-,ntrapping+ +1-5 K'.K-K

FIG. 2. Map of electron flow, and energy levels of chlorophyll for V of 0.3 V. Electron transfers are labeled e. Hole transfers are labeled o. The redox potential between inside and outside can be more than 2 V. The electrical potential between inside and outside is small. rophyll to the trap, and then a fast reaction transfers a hole from know that the decay of delayed light is much the same when the ch--ch* level of the outside chlorophyll to the trap. These the electron has been stopped. two fast reactions take turns. Two reactions are used to move Let R be the rate of the process that discharges the condenser an electron from inside to outside. As this alternating process in the absence of electron flow to the Calvin cycle. Assume that continues, a charge +Q is built up in the inside chlorophyll, and thermal fluctuations lift a hole from the ch-ch+ level of the a charge -Q is built up in the outside chlorophyll. inside chlorophyll to the still more positive level K-K + of some The charging of the little condenser gives a voltage across the molecule K that is inside the membrane. I think that K may be membrane that makes the four levels of the inside chlorophyll fl-carotene. [Stanier suggested that carotene protects the chlo- move to more positive values while the levels of the outside rophyll at high light intensities (12).] Next, an electron is chlorophyll move to more negative values. Let V stand for the transferred from the ch--ch level of the outside chlorophyll shift of the levels in volts. to the K*-K + level of carotene to make excited carotene. Be- In the light, the value of V increases until the overvoltage for cause carotene is nonfluorescent, the energy is converted into oxidation of water and the reduction voltage of the Hill oxidant heat. The whole process moves an electron from the outside to being used are reached and electron flow can start or until a the inside. The K process is a diode that prevents the voltage protective process described later intervenes. Fig. 2 shows the on the small condenser from becoming too large. map of electron flow when V has the value 0.3 V. By putting voltage on a sample of , I have been able to measure fluorescence and delayed light at the same time Number of electrons in Q (13). I find the intensities of fluorescence to be 143 times the The capacity of biological membranes is generally found to be intensity of delayed light. From that I take R to be 143 times 0.5-2.0 X 10-6 farads/cm2. If the area of one PSU is assumed S. Of course the level calculated is subject to revision when the to be (150 A)2, then the charge, Q, on the small condenser for ratio is better known. 0.6 V will be 4.2-16.8 electrons. The rate R is given by Delayed light R = Feexp -[(Vl - V)/kt]l [2] I assume that delayed light is made by thermal fluctuations where VI is the difference between the level K-K+, and 0.95 lifting an electron from the trap to the ch*-ch+ level of the the ch-ch+ level in the relaxed plant. inside chlorophyll, and lifting a hole from the trap to the For F as in Eq. 1 and for R to be 143 times S, VI must be ch--ch* level in the outside chlorophyll. Then the intensity of 0.676 V. Therefore, the K-K + level is at +1.626 V on the redox the delayed light, S, is given by the equation scale. To calculate the decay of delayed light in the dark and in the S = F expl-[(0.8 - V)/kT]l = G exp(40V) [1] absence of electron flow to the Calvin cycle, I let N be the number of electrons in the charge Q. where F is the frequency factor, about 109 sec1, and kT is V = JN about 1/40 eV at room temperature. Strictly speaking, the equation should contain the probability that the few chlorophyll where J is the proportionality factor; the differential equation molecules next to the trap contain a hole or an electron, but that will be factor will be ignored to make things simpler. dV/dt = J(dN/dt) = -JR = -JH exp(40 V) [3] The decay of the delayed light depends on the decay of V. For the delayed light to change by 106 times, V must change where H is 143 times G. by about 0.345 V. Integration gives Several experiments tell us that delayed light is not the main 1 process in the decay of V. The electron flow to the Calvin cycle exp(40 V) = 40 JHT + exp(-40 V0) [4[4] is only part of the process. When V is large, the electron flow is too small, and when V is so small that we do not have the where t is the time in the dark and VO is the value of V at time overvoltage, the electron flow is probably zero. Besides, we zero. Downloaded by guest on October 1, 2021 Botany: Arnold Proc. Natl. Acad. Sci. USA 73 (1976) 4505 Let I be the intensity of the exciting light and AI be the rate Conclusions that electrons are transferred by the fast reaction. Then in the The good things about this map of the electron flow are: (i) it steady state explains how light energy is converted to chemical energy. (ii) AI = H exp(40 V0) It gives the overvoltage and the span of redox potentials needed. = (iii) It explains the origin, the decay curve, and the saturation exp(-40 V0) H/AL. [5] of delayed light. (iv) It explains how carotene can protect From Eqs. 1, 4, and 5 the delayed light is chlorophyll at high light intensities. The bad things are: (i) it does not explain that part of the S= 1/143k40 JAIt + i) [6] delayed light due to recombination (15). (ii) It makes the glow curves more difficult to understand (16). The idea that there are three light reactions in photosynthesis Eq. 6 gives the right decay of delayed light with time, and has been used by Arnon et al. (17) in a paper that gives addi- shows how the light saturation of delayed light depends on the tional evidence for the map. time in the dark. A complete treatment would have to contain the probabilities This research was supported by the U.S. Energy Research and De- that I omitted and allow for the fact that when there is electron velopment Administration under contract with the Union Carbide flow the Q on the inside chlorophyll need not be the same as Corp. that on the outside. 1. Basshem, J. A. & Calvin, M. (1952) Path of Carbon in Photo- The two Qs will adjust so that the rate at which holes go to synthesis (Prentice-Hall, Inc., Englewood Cliffs, N.J.). oxidation of water is the same as that at which electrons go to 2. Hill, R. (1939) Proc. R. Soc. London Ser. B 127, 192-210. the Calvin cycle, and so that the sum of the rate of the electron 3. Emerson, R., Chalmers, R. & Cederstrand, C. (1957) Proc. Nati. flow and the rate R is equal to the rate at which light transfers Acad. Sci. USA 43,133-143. electrons from the inside chlorophyll to the outside chloro- 4. Duysens, L. N. M., Amesz, J. & Kamp, B. M. (1961) Nature 190, phyll. 510-514. 5. Weaver, E. C. & Weaver, H. E. (1969) Science 165,906-907. 6. Strehler, B. & Arnold, W. (1951) J. Gen. Physiol. 34, 809-820. Enhancement 7. Azzi, J. R. (1966) Oak Ridge National Laboratory Report TM- 1534. The oxidation of water inside the grana disks will produce 8. Nelson, R. C. (1968) Photochem. Photobiol. 8, 441-450. protons as well as 02. If we assume that the flow of protons out 9. Arnold, W. & Azzi, J. R. (1968) Proc. Natl. Acad. Sci. USA 61, of the disk makes, by the Mitchel reaction, some, but not all, of 29-35. the ATP that is needed for the Calvin cycle (14), then the extra 10. Joliot, P., Joliot, A., Bouges, B. & Barbieri, G. (1971) Photochem. ATP must be made by the flow of electrons from ferredoxin to Photobiol. 14, 287-305. cytochrome f. 11. Kok, B., Forbush, B. & McGloin, M. P. (1971) Photochem. Pho- The mixture of excitations of system I and system II where tobiol. 14, 307-321. the electron flow from system I can just make the extra ATP 12. Stanier, R. Y. (1959) Brookhaven Symp. Biol. 11, 43-53. 13. Arnold, W. (1972) Biophys. J. 12, 793-796. needed will give the best quantum yield. 14. Mitchell, P. (1966) Biol. Rev. 41, 445-502. If system I is not excited, then the extra ATP has to be made 15. Arnold, W. & Azzi, J. R. (1971) Photochem. Photobiol. 14, by the flow of expensive electrons (they cost two quanta) from 233-240. system II to ferredoxin to cytochrome f to 2. This will make 16. Arnold, W. (1966) Science 154, 1046-1049. for a smaller quantum yield. 17. Arnon, D. I., Knaff, D. B., McSwain, B. D., Chain, R. K. & Tsu- If only system I is excited we have no photosynthesis. jimoto, N. Y. (1971) Photochem. Photobiol. 14, 397-425. Downloaded by guest on October 1, 2021