ENERGY LEVELS AND ELECTRON FLOW IN * BY WILLIAM ARNOLD AND J. R. Azzi

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE Communicated July 16, 1968 At the Second International Conference on Photosensitization in Solids,' Dr. R. C. Nelson presented data on the absolute values of the energy levels of chlorophyll as a solid. These new values, together with measurements of "glow curves" that we have been making, allow us to give now a somewhat more real- istic picture of the operation of the photosynthetic apparatus in green plants. The work of Calvin and associates has shown2 that in the process of photo- synthesis, reduction is a series of reactions, none of which needs light. This reduction cycle (the ) is driven by electrons at -0.4 v and by ATP. The discovery by Hill' that could produce in the light if they had an electron acceptor present makes it possible to study the photoreaction part of photosynthesis divorced from carbon reduction. We now know that chloroplasts in the light produce ATP and electrons at -0.4 v, just as they should if they are to drive the Calvin cycle. Over the years since the discovery of the Hill reaction, the work of a great many different people has led to a theory as to how the photosynthetic apparatus works. This theory is known colloquially as the "Z-scheme." It started from a suggestion of Hill and Bendall4 and is elaborated in its most detailed form in scheme 6 of Witt's paper.' A simplified version of the Z-scheme is shown in Figure 1. Light absorbed by System II chlorophyll lifts an electron from the level of H20 at +0.8 v to 0 v, the electron then flows through an electron transport system to +0.4 v, and ATP is produced by this flow. (Two kinds of chlorophyll are required by the phe- nomenon of enhancement,6 and we use the nomenclature of Duysens,7 System I and System II, and the redox scale in which the reducing end is negative.) Light absorbed by System I chlorophyll lifts the electron from the level of f at +0.4 v to the level of at -0.4 v, from which it flows to the Calvin cycle. This theory has been useful in explaining many of the observations on photo- synthesis. Nevertheless, for other observations it is inadequate and we feel that it must be changed.

ELECTRONS TO -0.4- B THE CALVIN CYCLE FIG. 1.-A schematic representation for the _ Z-scheme, showing electron flow through Sys- ° o SYSTEM tem II to System I to the Calvin cycle with z V the production of ATP by the electron trans- I port chain. f+04 SYSTEM MJ\ XATP

+0.8 H20 29 Downloaded by guest on September 25, 2021 30o BOTANY: ARNOLD AND AZZI PROC. N. A. S.

We now give four objections to the Z-scheme: (1) Some time ago, it was found that green plants undergoing photosynthesis emit delayed light.8 This light has the same emission spectrum as the fluores- cence of chlorophyll in the living plant,9 but lasts far too long to be fluorescence. On the Z-scheme, since each quantum stores only +0.8 ev of the 1.83 ev of the absorbed quantum, presumably a thermal fluctuation must furnish 1 ev of energy. A simple calculation (using the frequency factor that we discuss below) shows that the Z-scheme predicts the intensity of the delayed light to be approximately 106 times too small. (2) Bertsch, West, and Hill10 have recently found that the delayed light from chloroplasts (measured 1 msec after illumination) is increased several times when ferricyanide is added to start the electron flow. When the delayed light. was first found, everyone assumed that photosynthesis (or electron flow, in the Hill reaction) must compete with delayed light for the stored energy. But this new observation suggests that two quanta cooperate to give both delayed light and electron flow. In order for the two quanta to be able to cooperate, they must be absorbed in the same chlorophyll system, not one in System I and the other in System II. (3) Jones'1 has shown that the intensity of delayed light made by a flash, on completely relaxed plants, is proportional to the square of the energy of the excit- ing flash. This means that two quanta cooperated to make the delayed light, and this is in agreement with the experiment of Bertsch et al. (4) We know that chlorophyll is the pigment that absorbs the energy used in photosynthesis. From the fact that chlorophyll in the plant fluoresces, we know that chlorophyll exists in the excited state for some time. It would seem that any theory of photosynthesis must involve the absolute energy levels of chlorophyll. This the Z-scheme does not do. Absolute Energy Levels of Chlorophyll. Nelson did not make his measurements on chlorophyll but rather on ethyl chlorophyllide a and b. Since there is no reason to expect any large difference in the levels, and since the precision of his measurement exceeds that of our glow curves, we will use his levels as if they were those of chlorophyll. He measured the energy necessary to extract an electron from the solid; for a metal, this would be called the work function. He also measured the electron affinity of the solid, that is, the energy given up by an electron to form chl- in the solid. In Figure 2, we show energy levels of chlorophyll as found by Nelson. We give both the absolute scale, where an electron at infinity in a vacuum is assigned zero energy, and the redox scale. To find the relation between the two scales, we used the absolute values -4.80 of the Fe++-Fe+++ level (given in Fig. 43 of Gurney's Ions in Solution'2). We compared this with the redox level (+0.77). A simple algorithm for obtaining the redox level is to add 4.00 to the absolute value and change the sign of the sum. Glow Curves.-Two years ago we showed that the method of glow curves,'3 used for many years to study the storage of energy in inorganic crystals, can also Downloaded by guest on September 25, 2021 VOL. 61, 1968 BOTANY: ARNOLD AND AZZI 31

ETHYL CHLOROPHYLLIDES -1.0- Chi a -2.96 --3.0 ChI o Chi -& -0.8- -3.1 Chlb* -3.2 Chi 6* -0.6- 3.3 --3.4 "-04~~~~~~~~ FIG. 2.-Nelson's energy levels for ethyl chlorophyllides a and b plotted against ,-Q2--. --3.8 both the redox scale in volts and the abso- X 0 --4.04 lute energy scale in electron volts. For the Cn0 transfer of one electron, the energy differ- O-4.2 0~~~~~~~~~~~~~~~~~~~, ence between two redox levels is in electron ca volts. r +0.4- --44O +0.6- -4.64

+0.8 Chl 0+ --4.8 +1.0- --5.0 Chi b+ +1.2- Chl b -5.16.6 --52 be used in the study of the light reaction in photosynthesis. Here we will give only a short description and some new results. A sample of green plant material (algae, leaf plugs, or chloroplasts) is held in the dark at room temperature until the effects of previous illumination have dis- appeared. We say that the sample is relaxed. The sample is frozen to some low temperature, between -10C and -196oC, illuminated, and heated at a constant rate from the low temperature to + 100'C. During the heating, a photomultiplier measures the light emission from the sample. With green plants five different light emissions are observed: four are spikes of emission that occur at certain temperatures during heating, and the fifth is a general emission between +30° and +100'C. The intensity of this light does not depend upon the rate of heating, as a glow curve should, but it does depend upon the presence of oxygen. In the glow curve, the four peaks of emission, which we have called Z, A, B, and C, occur at -155°, -6°, +30°, and +520C when we use a heating rate of 30/sec. We feel that the Z peak has nothing to do with photosynthesis, since one must use blue light to excite it and since it is present in plant material that had been previously heated at 1000C for 5 min. The three peaks A, B, and C give every indication of being intimately involved with the light reaction of photosynthesis. All three peaks disappear if the sample is heated to 550C for 5 min before making the experiment. In the presence of DCMU, only the B peak is found. Bishop's Scenedesmus Mutant #8, in which System I is inactive, gives all three peaks; Mutant #11, in which System II is inactive, gives none.'4 That the melting of the ice does not play an essential role is shown by the fact that we obtain the same three peaks, although at slightly different temperatures, when chloroplasts are suspended in 66 per cent glycerol with a melting point at -47°C. At the time the 1966 paper was written, we thought that the activation energy given by these glow curves represented the sum of the energy needed to return an electron to chlorophyll plus the energy needed to return a hole to chlorophyll Downloaded by guest on September 25, 2021 32 BOTANY: ARNOLD AND AZZI PROc. N. A. S.

Nelson's results make this unlikely, since we now know that an electron returned to chlorophyll goes to the chl- level, 0.2 ev above the level of excited chlorophyll, and that a hole returned to chl b+ level is 0.23 ev below the ground state of chloro- phyll a. This means that the activation energy for one of the peaks could be energy needed to return an electron to a photosynthetic unit that already has a hole, while the activation energy for another peak would be the energy needed to return a hole to a unit that contained an electron. We have no unambiguous argument as to which peak is electron-untrapping and which is hole-untrapping. By the addition of oxidizing or reducing sub- stances to chloroplasts, we find that oxidizing conditions tend to make the A and C peaks larger with respect to B. We also find that methyl viologen makes the B peak smaller. These observations suggest that the B peak is electron-untrap- ping and that A and C are hole-untrapping. If these peaks in light emission have been correctly identified as glow curves, they would be expected to follow the differential equation' NFeE/kT, (1) dtd= where N = the number of trapped electrons or holes, F = frequency factor, k = Boltzman constant, T = temperature in 'K, and E = the energy of activa- tion. The intensity of the emitted light is to be taken as proportional to dN/dt. The peak of light emission will occur when E eE/kT= FT (2) kT = 2 where $ = rate of heating in degrees per second. Randall and Wilkins'5 give 101 to 109 as the value of the frequency factor for untrapping electrons. Some time ago we estimated the frequency factor for dried chloroplasts and found 2.5 X 109.16 Using this value and equation (2), we find the activation energies for the three peaks to be 0.53, 0.60, and 0.64 ev. If we use the smaller value, 108, we find the activation energies for the three peaks to be 0.46, 0.52, and 0.56 ev. An Electron-Hole Picture ofPhotosynthesis.-At the First International Confer- ence on Photosensitization in Solids, one of us presented a paper,17 with the above title, in an attempt to explain the production of delayed light during photosyn- thesis. This theory was not satisfactory, in that it gave no reason why two light quanta should be required for System II. Nelson's new chlorophyll levels allow us to modify the picture so that it requires two quanta. It has been known for a long time that something like 2000 chlorophyll mole- cules are involved in producing one molecule of oxygen. Since this represents the transfer of four electrons, there must be 500 chlorophyll molecules in the photosynthetic unit that handles one electron; and the 500 has to be divided be- tween System I and System II. In Figure 3, the space labeled System II represents those of the 500 chlorophyll molecules that belong to System II. Chlorophyll a is on the right side of the unit and chlorophyll b on the left. Trap A, the reaction center on the right side, is Downloaded by guest on September 25, 2021 VOL. 61, 1968 BOTANY: ARNOLD AND AZZI 33

SYSTEM H SYSTEM I Chio ~~~~~~~~~~Chi0

-0.8-hi 'ChI C*hi o Chi b*I -0.6M FERREDOXIN -0.4- TRAP A le le TRAP A' M -J0. TRANSPORTiELECTRON CLIN CHAIN C,) ~~~WI psidea of Syste I W ATPII CYTOCHROME t 'r40.4 +0.6- oe Abou 500 Choohloeue r ob iie ewe yCLemIanIIfrec r0.8nH2o02TRAP B Chi a 0 Chi a+ +1.0- HiO CIb + 1.2

FIG. 3.-Chlorophyll energy levels and electron flow in the photosynthetic unit. About 500 chlorophyll molecules are to be divided between Systems I and II for each photosynthetic unit. In System I, only chlorophyll a energy levels are used. In System II, both chlorophyll a and b energy levels are used, with an interface between the two . System II uses two light quanta to take an electron from the level of H20 to the level of ferredoxin. System I uses one quantum to transfer an electron from the cytochromef level to ferredoxin. It is conceivable that System I is actually the chloro- phyll a side of System IL.

ferredoxin or is very nearly at the redox level of ferredoxin,e0.42.18 Trap B, the reaction center on the left, is shown at 0.815, the H20-02 potential. We suppose traps A and B to be on opposite sides of the photosynthetic unit so as to separate the reduction and oxidation. When the chlorophyll of System II absorbs a light quantum, it forms an ex- citon that can run over the whole system. This exciton can react at trap A to put an electron in the trap and leave a free hole in chl a+. The exciton cannot react with trap A if an electron is already there. Similarly, at trap B, an exciton can put a hole in the trap and leave an electron in chi b-. We further must postu- late that an electron can move through the chl b, probably by tunneling,' while a hole can move through the chl a+ level. But due to the mismatch of the energy levels, it is impossible, or very nearly impossible, for an electron to tunnel from chi b- to chi a- or for a hole to tunnel from chi a+ to chl b+. However, it is pos- sible for the chl b- and chi a+ to recombine, form excited chlorophyll, and thus give delayed light. The movement of electrons and holes gives the electronic conductor that must exist between traps A and B if the acts of oxidation and re- duction are to be separated spatially, thus preventing back reactions. Although it can be argued that this is an: illogical way to use the chlorophyll levels, we must remember that the photosynthetic machinery was not designed by~jogic, but by evolution. It could well be that green plants had been perform- Downloaded by guest on September 25, 2021 34 BOTANY: ARNOLD AND AZZI PROC. N. A. S.

ing photosynthesis for many millions of years using System I alone (as the purple bacteria do) before any of them realized that by inventing chlorophyll b, with its much more positive chl b+ level, they could extract electrons from . That is, the chlorophyll b part may have been added on to System I, in place of cytochromef, to form System II. The value 0.60 ev, for the activation energy of the B peak, the one that we think is electron-untrapping, agrees with the expected value of 0.59 ev for the difference between the levels of chl a- and ferredoxin. On the oxygen side, we do not have agreement. We expect one activation energy, at 0.35 ev. We have two, at 0.53 and 0.64 ev. We must remember that although +0.815 is the thermodynamic value, in fact it takes four holes to oxidize water to oxygen, and there is no reason to expect them all to have the same activa- tion energy. At the meeting in Tucson, Joliot'9 reported that although relaxed chloroplasts can give out electrons when illuminated by a single short flash of light, no oxygen is produced by one flash; but oxygen is produced by two flashes separated by a few milliseconds. This experiment (Franck had observed the oxygen aspect of the experiment long ago2O) shows that the oxygen side has at least two steps. The right side of Figure 3 shows System I chlorophyll. Here we suggest that the exciton can put an electron in the trap A' reaction center, leaving a hole in chl a+. The hole can "float up" and oxidize cytochromef. Since in System I we can have neither an electron nor a hole left, we expect no glow curve with Scene- desmus Mutant #11, as we found. It seems likely that the System I chlorophyll is identical with the right side of System II. The photosynthetic unit is a solar battery with three terminals, a negative terminal at the level of ferredoxin and two positive terminals, one at the level of cytochromef and one at the level of H20-02. .-For every electron that moves through System II from the B reaction center to the A reaction center, there must be a hydrogen ion diffusing from B to A. If these two centers are on opposite sides of a membrane and if the Mitchell hypothesis of ATP formation2' is correct, then hydrogen ion flow would give the noncyclic photophosphorylation using Arnon's nomenclature. Cyclic photophosphorylation involves the flow of electrons through System I and back through the electron transport chain, which we believe to be located between ferredoxin and cytochrome f. In pseudocyclic photophosphorylation, the electrons flow from water through System II, then through the electron trans- port chain to cytochrome f, and then to oxygen. Arnon has shown that ferre- doxin is involved in all three ways of making ATP."1 Discussion.-There are objections to the theory proposed in this paper, such as (1) if, for two quanta absorbed by System II, there is formed one excited chloro- phyll by recombination, then the ratio of delayed light to fluorescence should be approximately one half; however, the one measurement of this ratio by Lumry and Muller gave 0.08;22 (2) chlorophyll b plays an important role in the theory, but there are plants that perform photosynthesis and produce oxygen yet do not contain chlorophyll b; (3) Bishop found that chloroplasts made from Scenedesmus Mutant #8 (where System I is inactive) were unable to reduce NADP+ 2 The Downloaded by guest on September 25, 2021 VOL. 61, 1968 BOTANY: ARNOLD AND AZZI 35

chloroplasts should have made this reduction if reaction center A is actually at the level of ferredoxin. Nevertheless, the theory that we have sketched does give a mechanism for the production of delayed light and a simple explanation for the experiments of Jones'I and Bertsch et al.'0 Furthermore, the theory allows us to understand the energy storage in the photosynthetic unit observed in glow curves. It also furnishes an explanation for the phenomenon of enhancement. Perhaps the most important point of this theory is that it provides in a natural way an electronic conductor between the site of oxidation and the site of reduc- tion. This spatial separation would prevent back reactions and would allow us to understand the high efficiency with which green plants use light. Abbreviations: ATP, adenosine 5'-triphosphate; NADP+, nicotinamide-adenine dinucleo- tide , reduced form; chl, chlorophyll. * This research was sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. 1 Nelson, R. C., Second International Conference on Photosensitization in Solids (1968), to be published in Photochem. and Photobiol. 2 Bassham, J. A., and M. Calvin, The Path of Carbon in Photosynthesis (Englewood Cliffs, N. J.: Prentice Hall, 1952). 3 Hill, R., Proc. Roy. Soc. (London), 127, 192 (1939). 4Hill, R., and D. Bendall, Nature, 186, 136 (1960). 5 Witt, H. T., in Nobel Symposium V (New York: Interscience Publishers,1967), p. 291. 6 Emerson, R., R. Chalmers, and C. Cederstrand, these PROCEEDINGS, 43, 133 (1957). 7Duysens, L. N. M., J. Amesz, and B. M. Kamp, Nature, 190, 510 (1961). 8 Strehler, B., and William Arnold, J. Gas Physiol., 34, 809 (1951). 9 Azzi, J. R., Oak Ridge National Laboratory Technical Memo No. 1534, 1966. 10 Bertsch, W., J. West, and R. Hill, submitted to Biochim. Biophys. Acta. 11 Jones, Larry W., these PROCEEDINGS, 58, 75 (1967). 12 Gurney, R. W., Ions in Solution (London: Cambridge University Press, 1936). 13 Arnold, William, Science, 154, 1046 (1966). 14 Bishop, Norman I., Record Chem. Prog., 25, 181 (1964). 15 Randall, J. T., and M. H. F. Wilkins, Proc. Roy. Soc. (London), 184, 372 (1945). 16 Arnold, William, and Helen Sherwood, J. Phys. Chem., 63, 2 (1959). 17 Arnold, William, J. Phys. Chem., 69, 788 (1965). 18 Arnon, D. I., in A Symposium on Non- Iron Proteins: Role in Energy Conversion, ed. Anthony San Pietro (Yellow Springs, Ohio: Antioch Press, 1965), p. 137. 19 Joliot, P., Second International Conference on Photosensitization in Solids (1968), to be published in Photochen. and Photobiol. 20 Allen, F. L., and J. Franck, Arch. Biochem. Biophys., 58, 510 (1955). 21 Mitchell, P., Biol. Rev. Cambridge Phil. Soc., 41, 445 (1966). 22 Muller, Alexander, and Rufus Lumry, these PROCEEDINGS, 54, 1479 (1965). 23 Pratt, Lee H., and Norman I. Bishop, Biochim. Biophys. Acta, 153, 664 (1968). Downloaded by guest on September 25, 2021