path of electrons in photosynthesis

Path of Electrons in PhotosynthesisAuthor(s): William ArnoldSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 73, No. 12 (Dec., 1976), pp. 4502-4505Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/66143 .

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Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4502-4505, December 1976 Botany

Path of electrons in photosynthesis (energy levels of chlorophyll/delayed light/semiconductors/carn

WILLIAM ARNOLD

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Contributed by William Arnold, September 20, 1976

ABSTRACT Electrons, from the oxidation of water, inside the grana disks (thylakoids) are transferred across the membrane to the outside, to the Calvin cycle or the Hill oxidant. The span 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 electron trap is embedded in the membrane. Alternately, an excited chlorophyll on the inside gives an electron to the trap, and an excited chlorophyll on the outside gives a hole to the trap. Two quanta move an electron from inside to outside. The charging of this condenser drives the redox levels on the inside positive and those on the outside negative. The final voltages depend upon the electron flow and a carotene diode. A voltage of 0.3 is involved. Delayed light is the exact reverse of the light reaction. System I makes ATP.

Photosynthesis is the process by which green plants reduce CO2 to carbohydrates and oxidize water to 02. This process makes the food we eat and the 02 we breathe, and in past times made the coal and oil we now use. Energy, which comes from sunlight absorbed by chlorophyll, is stored to the extent of some 5.1 eV per carbon atom.

Research with radioactive carbon has made it possible to follow "The Path of Carbon in Photosynthesis" in detail (1). CO2 does not take part in a photochemical reaction. Carbon reduction is a series of enzyme reactions known as the Calvin cycle. This cycle can take place in the dark, and is driven by electrons at -0.4 V and by ATP. The chlorophyll apparatus (in the chloroplasts) must be able to use light energy to oxidize water to 02, lift electrons from the level of water (+0.8 V) to -0.4 V, and make ATP.

Exactly how the chlorophyll apparatus works is not known. Many attempts to explain its operation have been published, but none, including two that I have written, are satisfactory.

The present paper gives a new map for the flow of electrons in the chlorophyll apparatus. The paper is not a complete theory of photosynthesis, but an attempt to explain how the energy of light, absorbed by chlorophyll, is made available for chemical reaction.

There are four major constraints that we use in drawing the map.

Energy Levels of Chlorophyll. It has been known since the time of Stokes that the chlorophyll in green plants fluoresces. It is known that the intensity of the fluorescence depends on the rate of photosynthesis. We assume that the excitation of chlorophyll to the singlet state (1.8 eV) is the first step in photosynthesis.

The Hill Reaction: The Span in Redox Potential. Hill discovered that chloroplasts could be removed from plant cells and made to produce 02 in the light if they were provided with some reducible substance (2). These Hill oxidants seem to take the place of the entire Calvin cycle. Many Hill oxidants are now known, covering a wide range in redox levels; some are much more negative than the -0.4 V of the Calvin cycle, extending to about -1.0 V.

Abbreviation: PSU, photosynthetic unit.

451

>tene diode/system I and II)

The redox level for the oxidation of water to 02 is at +0.815 V. It is generally found that to make the reaction go, an overvoltage of about 0.5 V is needed.

The chlorophyll apparatus must be able to lift an electron from +1.3 to -1.0 V, a total span of about 2.3 V.

Enhancement. Emerson discovered that with monochro- matic light, as one goes through the spectrum the 02 production goes to zero at a shorter wavelength than the absorption of chlorophyll does (3). That is, some of the longer wavelength light absorbed by chlorophyll cannot give 02. Nevertheless, the mixing of this non-02-producing light with light that can produce 02 gives a higher quantum yield than either light alone.

This unexpected and baffling phenomenon, now known as "enhancement," has been the subject of much research since its discovery. It is believed that enhancement shows that there are two photochemical reactions in green plants (4). The long-wavelength (6850 A; 1.81 eV) reaction, system I, does not produce 02, and from the measurement of Weaver has about 110 chlorophyll molecules in the unit (5). System II, the short- wavelength (6700 A; 1.85 eV) reaction, does produce 02, and from general arguments about photosynthetic units must have about 500 chlorophyll molecules.

In a number of the attempts to explain the operation of the chlorophyll apparatus, the assumption has been that system II and system I are connected in series for the electron flow. Sys- tem II has 4.5 times as many chlorophyll molecules as system I. So an additional assumption has to be made-that system II can transfer excitation to system I-for the rate of electron transfer to be equal in the two systems. The energy difference (0.04 V) between the two systems is not large enough to prevent the transfer of excitation energy from system I to system II. So we should expect to see 02 production [exp(-0.04/kT) = 0.2] at wavelengths where only system I can absorb light. This we do not see.

In this paper, it is assumed that system II and system I are not in series.

Delayed Light. After illumination, green plants emit, in the dark, a dim glow that we call delayed light (6). The emission spectrum of this light is the same as that for the fluorescence of chlorophyll in the plants (7). This means that excited chlorophyll must be regenerated in the dark. After bright illumination, the intensity of delayed light is proportional to the re- ciprocal of the time in the dark, over the range of a few milli- seconds to 11/2 hr.

The intensity of the delayed light, at time t in the dark, be- comes saturated with respect to the intensity of the exciting light. That is, we can find an exciting intensity beyond which there is no increase in the delayed light. If t is small, the intensity of exciting light to give saturation is very high. If t is large, the intensity of exciting light to give saturation is very low.

Experiments show that most, if not all, of the delayed light

)2



Botany: Arnold

InsidE ch--ch Oxidi

C O- 1.8 eV 1.8eV 15C x 0

ch - ch+

Lipid (insula'

FIG. 1. Left side shows the four levels of chlorophyll on the redox , PSU.

is made by system II. Experiments also show that the delayed light is much the same in chloroplasts in which the electron flow has been stopped by poisons.

The fundamental assumption of this paper is that delayed light is made by the reverse of the fast reaction that stabilizes the energy of excited chlorophyll so that it can be used for photosynthesis.

Energy levels of chlorophyll

To draw the map of the flow of electrons in the chlorophyll apparatus, I must plot the various chlorophyll levels on the redox scale. Nelson measured the ionization energy and the electron affinity of solid ethyl chlorophyllide (8). In a later paper, Azzi and I used his data to estimate where the chlorophyll levels are on the redox scale (9). These values will be used here.

Nelson emphasized that if excited chlorophyll is the first actor in photosynthesis and if only one electron at a time is transferred, then there are four different redox levels.

The four levels will be denoted with symbols like "ch--ch," which means that a chlorophyll molecule with an extra electron can give the electron to some substance and reduce it, thereby becoming a neutral chlorophyll molecule. The left term in the symbol shows the chlorophyll when the level is filled, the right when the level is empty.

The symbol ch-ch+ denotes an oxidizing level. A chlorophyll molecule that has a positive charge (that is short one electron) can become neutral chlorophyll by giving up a hole (taking an electron) and thus oxidizing something.

Excited chlorophyll can act as a reducing level, ch*-ch+ (ch* - excited chlorophyll), by giving up the electron that has been promoted. The electron transfer must be very fast so as to compete with fluorescence.

A simple calculation shows that the electron transfer must be to a level 0.5-0.6 V more positive to allow enough time for chemical reaction. On the map this electron transfer is labeled as fast. After the fast transfer from the ch*-ch+ level, the oxidizing level ch-ch+ is left.

Excited chlorophyll can also act as an oxidizing level ch-- ch*. Crudely, we can think of the neutral excited molecule as having an electron in a high energy state and a hole in the ground state. The excited chlorophyll can take an electron (give up a hole) to fill the ground state and leave ch-. Again this must be a very fast reaction with an energy difference of about 0.5 V. This reaction is labeled as fast on the map.

Proc. Natl. Acad. Sci. USA 73 (1976) 4503

i Outside ing Iff _____ Reducing

A JK QTRAP

%--___?_ -Protein ^^ - t 1 ,(insulator)

Chlorophyll lembrane- (250 molecules) or)

,cale. Right side is a schematic of a possible cross section of system II

After the reaction the reducing level ch--ch is left. The transfer of electrons from this level is labeled as slow.

Fig. 1 (left) is a plot of the four levels on the redox scale.

System I

It is generally believed that the illumination of system I reduces ferredoxin (-0.4 V) and oxidizes cytochromef (+0.4 V). A fast transfer of an electron from the ch*-ch+ level (-0.85 V) to ferredoxin, followed by a slow transfer of a hole from the ch-ch+ level (+0.95 V) to cytochrome f, will do nicely.

System II

I think that the system II photosynthetic units (PSUs) are located on or in the membranes of the grana disks. Each disk is a small vesicle (thylakoid) shaped like a coin some 0.5 AM in diameter. If we take the diameter of one PSU to be 150 A, then there will be room for about 800 PSUs on each face of the disk. I think that the oxidation of water to 02 takes place inside of the vesicle. The system II PSUs transfer electrons across the membrane, from inside to outside, when the electrons are used by the Calvin cycle or the Hill reaction. Fig. I (right) is a possible cross section of a system II unit.

I assume that some of the 500 chlorophyll molecules are on one side of the membrane and some are on the other. Light energy absorbed by one chlorophyll can travel by resonance transfer to any other. This means that energy can flow freely across the membrane. Furthermore, some fractions of chlorophyll molecules on each side can act as semiconductors, and transfer electrons or holes from one molecule to the next. I also assume that the membrane is a good electrical insulator, as is the protein between the chlorophyll and the water on each side.

The square JK is the mechanism that oxidizes water, that Joliot and Kok are now studying (10, 11). Q is the substance that takes electrons from system II. T is an electron trap that is in the membrane.

Operation of system II-the map

Assume that in completely relaxed plants the four redox levels for the chlorophyll inside and outside are as shown in Fig. 1

(left), and the redox level of the trap is in the middle at -0.05 V.

Assume that when the system is illuminated, the fast reaction transfers electrons from the ch*-ch+ level of the inside chlo-



4504 Botany: Arnold

Inside Protein Chlorophyll Lip Oxidizing (insulator) (insL

+Q

-1.5

-1.0- LJ

-0.5 ?

x M o 0

rophyll to the trap, and then a fast reaction transfers a hole from

he

shift of the overlsvoltale? chls.ch

In the light, the value of V increases until the overvltage for +1.5- 0

FIG. 2. Map of electron flow, and energy levels of chlorophyll for V The redox potential between inside mbranes is generallyn be more than 2 be

rophyll to the trap, and then a fast reactionderansfers a hole from the ch0.6ch* level of the outside chlorophyll to the trap. These two fast reactions take turns. Two reactions are used to move an electron from inside to outside. As this alternating process continues, a charge +Q is built up in the inside chlorophyll, and a charge -Q is built up in the outside chlorophyll.

The charging of the little condenser gives a voltage across the membrane that makes the four levels of the inside chlorophyll move to more positive values while the levels of the outside chlorophyll move to morake negative values. Let V stand for the shift of the levels in volts.

In the light, the value of V increases until the overvoltage for oxidation of water and the reduction voltage of the Hmll oxidant being used are reached and electron flow can start or until a protective process described later intervenes. Fig. 2 shows the map of electron flow when V has the value 0. 3 V.

Number of electrons in Q The capacity of biological membranes is generally found to be 0.5-2.0 X 106 farads/cm2. If the area of one PSU is assumed to be (150 A)2, then the charge, Q, on the small condenser for 0.6 V will be 4.2-16.8 electrons.

Delayed light I assume that delayed light is made by thermal fluctuations lifting an electron from the trap to the ch*-ch+ level of the inside chlorophyll, and lifting a hole from the trap to the chb-ch* level in the outside chlorophyll. Then the intensity of the delayed light, S, is given by the equation

S = F-exp[-[(0 - V)/kT][ = G-exp(40V) [1]

where F is the frequency factor, about 109 sec1, and kT is about 1/40 eV at room temperature. Strictly speaking, the equation should contain the probability that the few chlorophyll molecules next to the trap contain a hole or an electron, but that factor will be ignored to make things simpler.

The decay of the delayed light depends on the decay of V.

overvoltage, the electron flow is probably zero. Besides, we

Proc. Natl. Acad. Sci. USA 73 (1976)

d Membrane Chlorophyll Protein Outside ilator) (insulator) Reducing

-Q

U ch-- ch

Calvin Cycle TRAP or

Hill Oxidant

ch ch*

]pping

of 0.3 V. Electron transfers are labeled e. Hole transfers are labeled o. rhe electrical potential between inside and outside is small.

know that the decay of delayed light is much the same when the electron has been stopped.

Let R be the rate of the process that discharges the condenser in the absence of electron flow to the Calvin cycle. Assume that thermal fluctuations lift a hole from the ch-ch+ level of the inside chlorophyll to the still more positive level K-K + of some molecule K that is inside the membrane. I think that K may be /-carotene. [Stanier suggested that carotene protects the chlorophyll at high light intensities (12).] Next, an electron is transferred from the ch--ch level of the outside chlorophyll to the K*-K + level of carotene to make excited carotene. Be- cause carotene is nonfluorescent, the energy is converted into heat. The whole process moves an electron from the outside to the inside. The K process is a diode that prevents the voltage on the small condenser from becoming too large.

By putting voltage on a sample of chloroplast, I have been able to measure fluorescence and delayed light at the same time (13). I find the intensities of fluorescence to be 143 times the intensity of delayed light. From that I take R to be 143 times S. Of course the level calculated is subject to revision when the ratio is better known.

The rate R is given by

R = F-exp[-[(Vl - V)/kt]} [2]

where V1 is the difference between the level K-K +, and 0.95 the ch-ch+ level in the relaxed plant.

For F as in Eq. 1 and for R to be 143 times S, V1 must be 0.676 V. Therefore, the K-K + level is at +1.626 V on the redox scale.

To calculate the decay of delayed light in the dark and in the absence of electron flow to the Calvin cycle, I let N be the number of electrons in the charge Q.

V = JN

where J is the proportionality factor; the differential equation will be

dV/dt = J(dN/dt) = -JR = -JH exp(40 V) [3]

where H is 143 times G.

Integration gives

exp(40 V) = 40 JHT + exp(-40 V,) [4]

where t is the time in the dark and V0 is the value of V at time zero.



Botany: Arnold

Let I be the intensity of the exciting light and AI be the rate that electrons are transferred by the fast reaction. Then in the steady state

AI- H exp(40 VO)

exp(-40 VO) = H/AI. [5]

From Eqs. 1, 4, and 5 the delayed light is

S=1/143(40 JAIt + 1) [6

Eq. 6 gives the right decay of delayed light with time, and shows how the light saturation of delayed light depends on the time in the dark.

A complete treatment would have to contain the probabilities that I omitted and allow for the fact that when there is electron flow the Q on the inside chlorophyll need not be the same as that on the outside.

The two Qs will adjust so that the rate at which holes go to oxidation of water is the same as that at which electrons go to the Calvin cycle, and so that the sum of the rate of the electron flow and the rate R is equal to the rate at which light transfers electrons from the inside chlorophyll to the outside chlorophyll.

Enhancement

The oxidation of water inside the grana disks will produce protons as well as 02. If we assume that the flow of protons out of the disk makes, by the Mitchel reaction, some, but not all, of the ATP that is needed for the Calvin cycle (14), then the extra ATP must be made by the flow of electrons from ferredoxin to cytochrome f.

The mixture of excitations of system I and system II where the electron flow from system I can just make the extra ATP needed will give the best quantum yield.

If system I is not excited, then the extra ATP has to be made by the flow of expensive electrons (they cost two quanta) from system II to ferredoxin to cytochrome f to 02. This will make for a smaller quantum yield.

If only system I is excited we have no photosynthesis.

Proc. Natl. Acad. Sci. USA 73 (1976) 4505

Conclusions

The good things about this map of the electron flow are: (i) it explains how light energy is converted to chemical energy. (ii) It gives the overvoltage and the span of redox potentials needed. (iii) It explains the origin, the decay curve, and the saturation of delayed light. (iv) It explains how carotene can protect chlorophyll at high light intensities.

The bad things are: (i) it does not explain that part of the 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 has been used by Arnon et al. (17) in a paper that gives additional evidence for the map.

This research was supported by the U.S. Energy Research and De- velopment Administration under contract with the Union Carbide Corp.

1L. Basshem, J. A. & Calvin, M. (1952) Path of Carbon in Photo- synthesis (Prentice-Hall, Inc., Englewood Cliffs, N.J.).

2. Hill, R. (1939) Proc. R. Soc. London Ser. B 127, 192-210. '3. Emerson, R., Chalmers, R. & Cederstrand, C. (1957) Proc. Natl.

Acad. Sci. USA 43, 133-143. 4. Duysens, L. N. M., Amesz, J. & Kamp, B. M. (1961) Nature 190,

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. 7. Azzi, J. R. (1966) Oak Ridge National Laboratory Report TM-

1534. 8. Nelson, R. C. (1968) Photochem. Photobiol. 8, 441-450. 9. Arnold, W. & Azzi, J. R. (1968) Proc. Natl. Acad. Sci. USA 61,

29-35. 10. Joliot, P., Joliot, A., Bouges, B. & Barbieri, G. (1971) Photochem.

Photobiol. 14, 287-305. 11. Kok, B., Forbush, B. & McGloin, M. P. (1971) Photochem. Pho-

tobiol. 14, 307-321. 12. Stanier, R. Y. (1959) Brookhaven Symp. Biol. 11, 43-53. 13. Arnold, W. (1972) Biophys. J. 12, 793-796. 14. Mitchell, P. (1966) Biol. Rev. 41, 445-502. 15. Arnold, W. & Azzi, J. R. (1971) Photochem. Photobiol. 14,

233-240. 16. Arnold, W. (1966) Science 154, 1046-1049. 17. Arnon, D. I., Knaff, D. B., McSwain, B. D., Chain, R. K. & Tsu-

jimoto, N. Y. (1971) Photochem. Photobiol. 14, 397-425.



path of electrons in photosynthesis

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