Photosynthesis

The Dark Reactions of Photosynthesis, Assimilation of Carbon Dioxide And The Calvin Cycle

C 3, C4 and CAM. Regulation of The Activity of Photosynthesis

The Light Reactions of Photosynthesis

The Photosynthetic Membrane Literature

The observation that a willow that has been cultivated in a container for five years with enough watering gained more than half a centner weight although only two ounces of the container's soil were lost goes back to J.B. van HELMONT (1577 - 1644). The British natural scientist S. HALES (1677 - 1761) understood that air and light are necessary for the nutrition of green plants. But it was not before the composition of air out of different gases became known that their significance for plant nutrition was studies. In 1771 observed J. PRIESTLEY (1733 - 1804), one of the discoverers of oxygen, that green plants give off oxygen and thus improve the air.

The priest J. SENEBIER (1742 - 1809) from Geneva discovered that the regeneration of the air is based on the use of 'fixed air' (carbon dioxide). These observations were confirmed and broadened by studies of the Dutch doctor J. INGENHOUSZ (1730 - 1799) who recognized both the meaning of light and the fact that the whole carbon contained in plants is of atmospheric origin. He, too, conceived that plants take up small amounts of oxygen at night or in the shadow and give off carbon dioxide. In 1804 discovered Th. des SAUSSURE (1767 - 1845) from Geneva that the plants' increase in weight cannot solely be caused by the uptake of carbon and minerals, but is based on the binding of the water components, too.

In 1894 constructed T. W. ENGELMANN (1843 - 1909) a gadget out of a modified microscope condenser that allowed him to expose parts of photosynthetically active cells (of the green alga Spirogyra) to a thin ray of light. His aim was to discover which components of the cell functioned as light receptors. To measure the oxygen production, he dispersed the thread-like Spirogyra in a bacteria-containing suspension. Whenever parts of the chloroplast were illuminated, did the bacteria concentrate in this area (where

oxygen was available). The illumination of other parts of the cell resulted in no such aggregations.

In an earlier study did he split white light into its spectral components using a prism. He then illuminated a green alga, Chladophora, with this spectrum. In contrast to Spirogyra are the Chladophora cells completely and evenly filled by the chloroplast. He observed that the bacteria accumulated mainly in the blue and red light. A first action spectrum of photosynthesis was thus yielded. It resembles roughly the absorption spectra of chlorophyll a and b.

J. v. SACHS (1832 - 1897) could finally prove that chlorophyll is involved in photosynthesis. In addition did he show that starch is produced in chloroplasts as a result of the photosynthetic activities.

These results are in accord with the first law of thermodynamics, whose discoverer J. R. MAYER postulated already in 1842 that plants take up energy in the form of light and that they transform it into another, a chemical state of energy. Based on this assumption was the reaction equation

6 carbon dioxide + 6 water > (chlorophyll) > glucose + 6 oxygen

formulated.

J.v. LIEBIG assumed that the oxygen stems from the breakdown of the carbon dioxide. This idea was uncritically accepted by the plant physiologists of the late 19th and the early 20th century (SACHS, PFEFFER, JOST and others) although M. J. SCHLEIDEN had as soon as 1842 realized that

1. glucose is produced as a result of photosynthesis (and he was closer to reality than SACHS was later) and that

2. it is very likely that it is water that is broken down. He wrote:

"It is well-known that CO2 is among the most stabile compounds and that no chemical way of breaking it down is known while H2O is very easily broken down.... and it does therefore seem likely that the 24 H2 of the 24 H2O are combined with the 12 CO2."

(from: Grundzüge der wissenschaftlichen Botanik). SCHLEIDEN's equations contain all reaction compounds in double numbers. He gives C12H24O12 as the formula for glucose.

It was soon realized that the reaction equation above is a simplification and that photosynthesis consists of a number of partial processes.

F. F. BLACKMAN and G. L. C. MATHGEL (1905, University of Cambridge, Great Britain) were among the first to study this topic systematically. They cultivated plants under different but controlled carbon dioxide concentrations, different light intensities and different temperatures and they noted the effects of these parameters on the rate of photosynthesis. Two decisive aspects were revealed. Under strong light and limited amounts of carbon dioxide is the rate of photosynthesis dependent on the temperature. This shows that the carbon dioxide fixation is based on normal, temperature-dependent biochemical reactions. Under carbon dioxide excess and too little light was no temperature-dependence found. This hints at the fact that the light-induced reactions are independent of the temperature. This statement applies to all photochemical reactions.

In 1925 put O. WARBURG (Kaiser-Wilhelm-Institut [later Max-Planck-Institut] für Zellphysiologie at Berlin-Dahlem) the results of BLACKMAN down to the existence of two classes of photosynthetic reactions: the light and the dark reactions.

During the thirties analyzed C. B. van NIEL (Stanford University) the photosynthesis of a number of purple bacteria. In addition to carbon dioxide do these bacteria need hydrogen sulphide for photosynthesis. van NIEL was able to determine

6 CO2 + 12 H2S > (light) > C6Hl2O6 + 12 S + 6 H2O

as the reaction's equation. Based on it did he extrapolate a general equation of photosynthesis:

CO2 + 2 H2X > (light) > (CH2O) + H2O + 2 X

According to this equation is photosynthesis a redox reaction with H2X as the electron donator (the oxydizable substance). In the case of green plants is it H2O and this means that not the carbon dioxide but the water is broken down.

A first experimental prove that the oxygen developed during the photosynthesis of green plants stems indeed from water was delivered by the British physiologist R. HILL. He detected that isolated chloroplasts give off oxygen in the presence of unnatural reducing agents like iron oxalate, ferricyanide or benzoquinone after exposure to light. The reaction went down in literature as the HILL-reaction:

2 H2O + 2 A > (light, chloroplasts) > 2 AH2 + O2

where A is the electron acceptor. If A = FeIII, then is

2 H2O + 4 FeIII > (light, chloroplasts) > 4 FeII + O2 + 4 H+

The process is linked to a photolytic breakdown of water that precede the reductionn of FeIII.

4 H2O > (light, chloroplasts) > 4 H+ + 4 OH-

This shows that

oxygen can also be set free in the absence of carbon dioxide,

the oxygen produced stems from the breakdown of water,

isolated chloroplasts are able to perform at least partial processes of photosynthesis.

The statement that the oxygen produced during photosynthesis stems only from the breakdown of water was confirmed by S. M. RUBEN, M. RANDALL, M. KAMEN and J. L. HYDE in 1941 after the isotope technique had found its way to biochemistry. They could shown that a suspension of Chlorella grown in H2

18O, gives off 18O2, after light exposure. Shortly afterwards confirmed S. M. RUBEN and his collaborators the postulate of O. WARBURG that the fixation of carbon dioxide is energy consuming but independent of light. In addition could E. RACKER (Cornell University, Ithaca, N. Y.) prove that light can be replaced by the addition of energy-rich compounds.

The Dark Reactions of Photosynthesis, Assimilation of Carbon Dioxide And The CALVIN Cycle.

Due to the use of isotopes were M. CALVN and his collaborators at the University of California, Berkeley able to reveal completely the reactions taking place during the incorporation of carbon dioxide into carbohydrates in the relatively short period from 1946 - 1953. The quick success was based on the use of sensitive methods (two-dimensional paper chromatography, autoradiography), a suitable specimen and the rapid progresses of enzyme biochemistry. Cultures of the single-celled green alga Chlorella pyrenoidosa (that was introduced to photosynthetic studies in 1919 by O. WARBURG) were supplied with light and an even stream of air containing 12CO2.

At a given time (t= 0) was 14CO2 added to the stream of air for a short time. It was assumed that the labelled carbon dioxide molecules were successively incorporated into intermediates of the carbohydrate synthesis. After 3, 5 etc. seconds were the experiments stopped by adding boiling alcohol and the newly produced 14C-labelled intermediates were separated and identified by paper chromatography.

1. The first stable compound that was labelled radioactively already after 3 seconds was 3-phosphoglycerate (3-PG), a substance we got to know previously as an intermediate of glycolysis. 14C is found in the carboxyl group of 3-PG. At first was it assumed that the molecule accepting the carbon dioxide would have to be a C2 unit. But after a futile search was finally ribulose diphosphate (RuDP), a C5 unit identified as the acceptor

C5 + C1 = 2 C3

This reaction is catalyzed by ribulose bisphosphate carboxylase (also called Rubisco or, formerly, fraction-1-protein), as far as quantity is concerned the most common protein of the world. The protein complex of green plants consists of eight times two subunits, eight large and eight small ones The picture to the right shows part of the enzyme together with ribulose phosphate , CO2 , and a magnesium ion (green ball) essential for the reaction. An interactive file demonstrates the single, subsequent reactions.

After longer reaction periods (5 seconds, 10 seconds, etc.) were further labelled compounds found. CALVIN and BENSON determined the sequence of the incorporation and were able to unite the single steps to a pathway. Two results were especially interesting:

o the resynthesis of ribulose diphosphate and o the production of the assimilate (the net product of the carbon dioxide

assimilation).

The production of ribulose diphosphate is best described by a cycle (the CALVIN cycle), while the assimilate production is a linear process. It is based on the fact that an intermediate of the CALVIN cycle is deducted from it.

2. 3-phosphoglycerate is reduced to glycerinaldehyde-3-phosphate (GAP), the carboxyl group is transformed into an aldehyde group. The reaction consumes ATP and NADPH2. The reverse reaction occurs, too, in glycolysis though in photosynthesis NADP is needed instead of the NAD consumed during glycolysis. It is known today that the two reactions (and all others, too) are catalyzed by different enzymes and that the enzymes of photosynthesis use NADP (> NADPH2) as a cofactor.

The CALVIN cycle has to be passed three times in order to produce one molecule of glycerinaldehyde-3-phosphate (a C<SUB<3< sub> unit) via photosynthesis since just one molecule of carbon dioxide is fixed in every round.

3. Just as in glycolysis is part of the glycerinaldehyde-3-phosphate converted into dihydroxyacetonephosphate (DAP) by epimerization.

4. Fructose-1,6-diphosphate (F-1,6-P) is formed by addition of one molecule glycerinaldehyde-3-phosphate and one molecule dihydroxyacetonephosphate.

5. Fructose-1,6-diphosphate is converted into fructose-6-phosphate (F-6-P) by splitting off Pi. The F-6-P has two alternative fates:

o One of the F-6-P molecules is converted into glucose-6-phosphate (G-6-P) that is sluiced away from the CALVIN cycle and is the net yield of photosynthesis.

o The other F-6-P disintegrates into a C5 unit (xylulose-5-phosphate; X-5-P) and a C1 unit that forms a C4 unit (erythrose-4-phosphate; E-4-P) together with GAP.

6. The E-4-P is coupled to one molecule of dihydroxyacetonephosphate (DAP). The result is a molecule of sedoheptulose-1,7-diphosphate (SDP), a C7 unit.

7. After splitting off one of the two phosphate residues reacts the sedoheptulose-7-phosphate (S-7-P) with glycerinaldehydephosphate. Two C5 units are the result: ribulose-5-phosphate (Ru-5-P) and xylulose-5-phosphate (X-5-P).

8. Ribulose-5-phosphate is phosphorylated to ribulose-1,5-diphosphate and starts a new round of the CALVIN cycle.

In summary, one can describe the end result of the dark reactions as follows: 6 RuDP + 6 CO2 > 12 3-PG 12 3-PG + 12 NADPH2 + 12 ATP > 12 GAP + 12 ADP + 12 Pi + 12 NADP

12 GAP > 1 glucose (net synthesis product of carbon dioxide assimilation) + 10 GAP 10 GAP + 6 ATP > 6 RuDP

NADPH2 and ATP stem, as we will see, from the light reactions of photosynthesis in which the light energy is converted into chemical energy.

After understanding the pathway in Chlorella pyrenoidosa arose the question whether it occurs in all other green plants, too. It could be shown to be an important pathway of all green plants. Even isolated chloroplasts (from spinach, for example) are still fully active and all reactions of the CALVIN cycle take part within them.

C3, C4 and CAM. Regulation of The Activity of Photosynthesis

C4

A number of plants display an increased and more efficient net photosynthesis during strong light intensities. A prime example are the Gramineae of warmer regions like maize or sugar-cane.

At the beginning of the sixties observed H. KORTSCHAK (Hawaiian Sugar Planter's Association) that the first product of photosynthesis in sugar-cane is not the C3 unit 3-phosphoglycerate but a unit with four C-atoms. The Australian plant physiologist M. D. HATCH and his English colleague C. R. SLACK confirmed this result and identified the compound as oxaloacetate (OAA). It is produced by the addition of one molecule of carbon dioxide to phosphoenolpyruvate (PEP). The cycle is also known as the HATCH- SLACK-cycle or the C4 cycle. Plants with this cycle are called C4-plants (and CAM plants, respectively) in contrast to C3 plants where the carbon dioxide is directly fed into the CALVIN cycle. The oxaloacetate is usually converted into malate of which the carbon dioxide is split off again with the help of an enzyme.

This carbon dioxide is now bound by ribulose-1,5-diphosphate and assimilated via the CALVIN cycle. :

Some species use malate instead of aspartate

oxaloacetate + L-glutamate > aspartate + alpha-ketoglutarate.

The reversible binding of carbon dioxide has the function to accumulate and store CO2. The process consumes energy, so that it could also be spoken of a carbon dioxide pump. It should be mentioned that the HATCH-SLACK cycle requires two molecules of ATP are per fixed carbon dioxide.

Photosynthesis of C4 plants. CO2 is bound to phosphoenolpyruvate (PEP) in mesophyll cells. The product is oxaloacetate. The next step generates malate. In the cells of the vascular bundle sheath, the 'Kranz' cells, is carbon dioxide split off the malate and fed into the CALVIN cycle. The pyruvate is transported back into the mesophyll cells (active transport) and is with the help of additional ATP phosphorylated to PEP.

The anatomy of C4 leaves with so-called 'Kranz' cells differs fundamentally from that of C3 plants. The chloroplasts of C3 plants are of homogeneous structure, while two types of chloroplasts occur in C4 plants. The mesophyll cells contain normal chloroplasts, that of the vascular bundle sheath have chloroplasts without grana , i.e. they are partially impaired in function. This peculiarity does not affect the CALVIN cycle, it concerns only the light reactions of photosynthesis. The first binding of carbon dioxide (the HATCH-

SLACK reaction) occurs in the mesophyll cells, the incorporation into carbohydrates (the CALVIN cycle) in the cells of the vascular bundle sheath. Both processes of photosynthesis are spatially separated.

The Crassulacean Acid Metabolism (CAM)

CAM is the abbreviation of Crassulacean acid metabolism. The name points at the fact that this pathway occurs mainly in Crassulacean species (and other succulent plants). The chemical reaction of the carbon dioxide accumulation is similar to that of C4 plants but here are carbon dioxide fixation and its assimilation not separated spatially but in time. CAM plants occur mainly in arid regions. The opening of the stomata to take up carbon dioxide is always connected with large losses of water. To inhibit this loss during intense sun (the transpiration via the cuticle remains intact) has a mechanism developed that allows the uptake of carbon dioxide during the night. The prefixed carbon dioxide is stored in the vacuoles as malate (and isocitrate) and is used during the daytime for photosynthesis.

Which Metabolism Goes With Which Conditions?

The enzyme that catalyzes the primary carbon dioxide fixation of C4 and CAM plants is phosphoenolpyruvate carboxylase (PEPC). Its affinity for carbon dioxide is by far higher than that of Rubisco, the first enzyme of the CALVIN cycle. As a consequence are C4 plants able to use even trace amounts of carbon dioxide. PEPC occurs in small amounts (roughly 2 - 3 %) also in C3 plants, where it, too, has a key position in the metabolic regulation.

Carbon dioxide yield of C4 and C3 plants of open grasslands in different parts of the world. In temperate regions is the rather low light intensity decisive for the disadvantage of C4 plants. C3 plants have an advantage due to their low rate of photorespiration and because they need no energy for the previous fixation of CO2. (J. R. EHRLICHER, 1978).

In growing roots (of maize seedlings, for example) does PEPC help to supply the lipid synthesis with NADH + H+. The following reactions take place:

1. phosphoenolpyruvate + HCO3- > oxaloacetate + Pi 2. oxaloacetate > malate. - During this step is NADH + H+ oxidized to NAD+. 3. malate > pyruvate + CO2.

During the last reaction is NAD reduced to NADH + H+.

In the root nodules of leguminosae is nitrogen fixed. Enough carbon bodies have to be supplied in order to incorporate the ammonia produced by the bacteria. the CO2-binding via PEPC-reaction thus is an important supplement

Furthermore produces the PEPC intermediates of the citric acid cycle (oxaloacetate and / or malate), a back-up in case of shortages. The activity of PEPC is controlled by extern factors, the day length is decisive. In some cases have different isoenzymes be found in different tissues. Their production is controlled by different triggers.

C3 plants can loose up to 20 percent of the carbon fixed in the CALVIN cycle at intense radiation. Under strong light is the photorespiration 1.5 - 3.5 times as high as the usual respiration in darkness. In C4 plants becomes the photorespiration drastically reduced, it may even not be detectable any more. In other words:

The net rate of photosynthesis (and consequently also the net production of biomass) of C4 plants is far larger at high light intensities than that of C3 plants. The optimal temperature of photosynthesis is below that of the respiration in darkness. As a consequence are losses caused by respiration larger at high than at low temperatures. Where light is a limiting factor and temperatures are low (i.e. in temperate climatic zones) have C3 plants the advantage, C4 plant do hardly occur (one of the exceptions is Spartina townsendii). C4 plants, nearly always herbs or shrubs, are more successful in the open country of warmer zones.

It has to be mentioned that two molecules of ATP are consumed in the HATCH-SLACK cycle

C4 plants belong to numerous, phylogenetically not related monocotyledonous and dicotyledonous families. Moreover have C4 activities also been detected in the blue-green alga Anacystis nidulans as well as in some dinoflagellates.

Since the alternative C3 or C4 is accompanied by considerable changes of the leaf anatomy has it to be assumed that the genetic potential for both pathways is quite common in the plant kingdom and that, depending on the ecological needs, one way is chosen by a species while a related species may choose the other one.

A well-studied example is the genus Atriplex, where both ways are realized. The C3 plants belong to one phylogenetic group, the C4 plants to another. In some cases can hybrids of C3 and C4 species be generated.

Influence of different parameters on the efficiency of the carbon dioxide uptake (ordinate) of a C3 plant (Atriplex patula, yellow line) and a C4 plant (Atriplex rosea, green line). Measured parameters (from left to right): light intensity, leaf temperature and concentration of carbon dioxide within the intercellular space (according to O. BJÖRKMAN and J. BERRY, 1973).

In several plant species of the genera Zea, Mollugo, Moricandia, Flaveria, etc. occur both types of CO2 fixation within one plant. In younger plants is usually the C3-, in older ones the C4 pathway taken. The amount of C4 is controlled by environmental factors.

CAM: Advantages and Disadvantages. CAM has been detected in more than 1000 angiosperms of 17 different families. It is usually accompanied by succulence, though not all Crassulaceae, for example, display CAM and succulence is no precondition of CAM. Tillandsia usneoides of the bromelia family is not succulent, but uses CAM. Mesemryanthemum crystallinum (a plant with succulent leaves) can use the C3 pathway but switches to CAM when growing in saline soils. Under experimental conditions can the shift be achieved by increasing the NaCl concentration of the nutrient medium (K. WINTER and D. J. von WILLERT, 1972). While the advantage of C4 plants comes in useful under high light intensities, is the degree of the CAM influence in CAM plants regulated mainly by temperature, atmospheric humidity and salinity. Both strong and weak CAM plants are known. In weak CAM plants becomes CAM only apparent at certain differences between day and night temperature. CAM plants that store a lot of malate and due to the thus high osmotic value also a lot of water, are usually less frost resistant than C3 plants. Because of the high concentration of acid are they less heat resistant, too. Species of arid regions are therefore forced to break their pool of malate down during the daytime (R. LÖSCH and H. KAPPEN, Universität Kiel, 1985). Usually do the C4 pathway and CAM exclude each other. An exception is the succulent C4

dicotyledon Portulaca oleracea that is able to choose the optimal pathway. under natural conditions

The Light Reactions of Photosynthesis

It has been mentioned in the historical outline that photosynthesis is dependent on light. The results of ENGELMANN and SACHS showed that it is absorbed by chlorophyll. We also got to know that plants have two types of chlorophyll, chlorophyll a and b and that both types display a characteristic absorption spectrum.

The action spectrum of photosynthesis resembles the absorption spectra of chlorophyll though it is not identical. This means that further photoreceptors (so-called accessory pigments) exist.

We already got to know something about the assimilation of carbon dioxide in the previous section. Our question is now: which reactions are induced by light and how is the light energy converted into chemical energy? Or, in other words, how are ATP and NADPH2 produced?

CALVIN and his collaborators studied the dark reactions in intact, active cells. This attempt proved to be insufficient for the light reactions. The results were contradictory. Techniques to isolate active chloroplasts had to be developed.

After the use of fractions containing isolated chloroplasts became usual, were three research groups at the same time (1951) and independent of each other able to show that isolated chloroplasts reduce NADP to NADPH2 when exposed to light [W. VISHNIAC and S. OCHOA (Rockefeller Institute, New York), L. J. TOLMACH (University of Chicago) and D. I. ARNON (University of California, Berkeley)]. Shortly afterwards (in 1954) discovered ARNON and his collaborators that the production of ATP, too, is dependent on light and that both ATP and NADPH2 can be produced simultaneously. Both compounds are generated from precursors that are present in the chloroplast already before photosynthesis starts, since no extern metabolites were supplied during the experiments. Accordingly was light (photons) the only available source of energy. It turned out that the production of ATP needs no oxygen, neither is oxygen produced during the reaction. Consequently runs the equation as follows:

n ADP + n Pi > (photons) > n ATP

The process is termed photophosphorylation. It exists in bacteria and blue-green algae, too, and is a general feature of photosynthetic processes.

After it was proven hat ATP is produced, was it asked how this is done. It seemed unlikely that the light induces the production of ATP directly but it had turned out that the production of ATP has to be preceded by an exposure to light. The concept of a light-induced electron flow was developed. It assumes that one molecule of chlorophyll

absorbs one photon. As a consequence is an electrons of chlorophyll transferred to a higher energy level.

This energy-rich electron is then transferred to a neighbouring electron acceptor with a strong electronegative redox potential. The transfer of the electron from the activated chlorophyll to the (first) acceptor is the first photochemical phase of photosynthesis. Its decisive feature is the transformation of a photon flow (light) into a flow of electrons.

As soon as a strongly electronegative (reducing) substance has been produced can the electron flow proceed with electron acceptors of less negative redox potentials. The process releases chemical energy that is used for photophosphorylation. Already during the fifties existed the first strong proves for the involvement of the chloroplasts' cytochromes. It could be shown, too, that the electron is finally accepted by a chlorophyll, so that its original state is restored again. The requirements of catalysis are fulfilled. The process became known as cyclic phosphorylation (D. I. ARNON, 1959).

Such a cyclic flow of electrons that is powered by light and releases chemical energy used for the production of ATP is unique. It is the outstanding property of photosynthetic cells.

Concept of cyclic photophosphorylation (to the left). To the right: the original concept of non-cyclic photophosphorylation (according to D. I. ARNON, 1971)

The only unexplained process remained was the photoreduction of NADP in chloroplasts. It were again ARNON and his collaborators that were in 1957 able to discover a second part of photophosphorylation. They could prove experimentally that the photoreduction of NADP and the synthesis of ATP are coupled. In contrast to the cyclic

photophosphorylation is the production of ATP coupled stoichiometric to a light-induced transfer of electrons from water to NADP and to the production of oxygen. The ATP production of the whole system increases the reduction rate of NADP. This pointed at the fact that the process is tightly coupled to cyclic photophosphorylation. Since electrons are irreversibly transferred from chlorophyll to NADP, are substitutes needed and these electrons stem from the breakdown of water. It is spoken of non-cyclic photophosphorylation, since ATP is produced simultaneously. Ferredoxin (a heme-less iron-sulphur protein) has a key position in this process. Its reduction potential is far more negative than that of NADP so that an electron flow from ferredoxin to NADP was very likely. The reduction of NADP is a three step reaction:

1. a photochemical reduction of ferredoxin that is followed by two 'dark' steps. 2. The re-oxidation of ferredoxin with the help of a ferredoxin-NADP-reductase (a

flavoprotein). 3. The re-oxidation of the ferredoxin-NADP-reductase by NADP.

     

Ferredoxin: Iron-sulphur-complex: - to the left: Fe2S2 protein - Planttype ferredoxins , to the right: Fe4S4 proteins - Bacterialtype ferredoxins

from: PROMISE - The Prosthetic groups and Metal Ions in Protein Active Sites Database

What was at first regarded as a photoreduction of NADP proved to be an electron transport chain that runs from ferredoxin via a flavin component to NADP.

The outstanding position of ferredoxin was strengthened even more after it was found out that stoichiometric amounts of O2 and ATP are produced during the reaction. The non-cyclic photophosphorylation can accordingly be described as follows:

4 ferredoxin (oxidized) + 2 ADP + 2 Pi + 2 H2O > (photons) > 4 ferredoxin (reduced) + 2 ATP + O2 + 4 H+.

The results led to the question how this reaction is coupled to the cyclic photophosphorylation discussed at the beginning. A series of experiments using specific inhibitors showed that ferredoxin is a component of that pathway, too.

So much about the chemical data. More knowledge about the primary effect of the light and about the significance of chlorophyll would have to exist to interpret them. These problems, too, have a long past history.

Two Photosystems

In 1932 exposed R. EMERSON and U. ARNOLD of the University of Illinois at Urbana Chlorella cells to a series of extremely short flashes of light. With this experiment did they try to find out how many molecules of chlorophyll were necessary to use one photon for the production of one molecule of oxygen. The result was that several hundred chlorophyll molecules are necessary which means that not all of them are of the same importance. Most act as light traps (or antennas) helping to transfer a photon to a reaction centre where an especially exposed chlorophyll transforms light energy into chemical energy. H. GAFFRON called this complex of several hundred chlorophyll molecules and other pigments (carotenes, carotenoids, xanthophylls, etc.) a photosynthetic unit.

This aggregation of pigments seems to lead to an especially efficient use of the incoming light. Still, a rather large part of the irradiated energy is lost. It does never reach the reaction centre and is emitted as warmth or light (red autofluorescence of chlorophyll).

When regarding the absorption spectrum of photosynthesis does it stand out that the efficiency of light of the wave length lambda > 680 nm decreases strongly although chlorophyll a displays an absorption in that area. R. EMERSON (1957) discovered that light of the wave length lambda > 700 (710) increases the rate of photosynthesis drastically if light of the wave length lambda = 680 nm (or less) is present at the same time.

When these two light qualities are used independent of each other or one after the other is no increase measured. EMERSON concluded that two photochemical processes have to exist that consist of different pigment systems (light receptors) but that do co-operate (EMERSON-effect). According to a suggestion of L. N.M. DUYSENS are the two systems called

photosystem I (PS I). It needs light of longer wave lengths (lambda > 700 nm) and

photosystem II (PS II). It becomes active when exposed to shorter wave lengths (lambda < 680 nm)

The ratio of chlorophyll a to chlorophyll b is higher in PS I than in PS II. The question how the two systems co-operate and how they are coupled to the production of ATP and NADPH2 remains to be settled.

ARNON and his collaborators could prove that the two systems are arranged in series and that both systems are required to explain all effects that had been recognized as photosynthetic ones. Only some bacteria that produce no oxygen during photosynthesis lack photosystem II. This results hints at the suggestion that the splitting of water is coupled to photosystem II and that photosystem I developed earlier in evolution.

The reaction centre of every photosystem is represented by one molecule of chlorophyll a each (P 700 in PS I and P 680 in PS II, where P means pigment).

The absorption of a photon by P 680 (which has a positive redox potential of + 0,8 V in its basic state) transfers P 680 into its excited state ( with a redox potential of 0,0 V) and causes the formation of a strongly oxidizing component (Z+) and a weakly reducing (Q-) one. Z+ withdraws electrons from water so that O2 and protons are set free.

4 Z+ + 2 H2O > 4 Z + 4 H+ + O2

The reducing component (a membrane-bound plastoquinone) feeds the electron into an electron transport chain in the course of which it looses energy part of which is used for the production of ATP. The electron does not return to its starting point (chlorophyll P 680) but is transferred to a chlorophyll molecule of photosystem I (P 700). The two photosystems are thus coupled.

The absorption of a further photon excites the P 700 just mentioned (the redox potential of which is + 0,4 - + 0,5 in its basic state). It transfers one electron to a membrane bound ferredoxin (P430) which passes the electron on to a soluble ferredoxin. The following steps are known.

In a stoichiometric sense is the outline above incomplete since ferredoxin transfers just one electron at any given time while two electrons are needed for the production of one NADPH2. The equation would consequently have to be:

2 ferredoxin (reduced) + 2 H+ + NADP+ > 2 ferredoxin (oxidized) + NADPH2

In summary is the light energy used for the flow of electrons from water to NADPH2 and for the simultaneous production of ATP (Z-scheme).

During our discussion did we neglect the cyclic photophosphorylation mentioned at the beginning. It proved to be a parallel process that starts work as soon as enough NADPH2 but too small amounts of ATP are present. Only photosystem I participates in cyclic photophosphorylation.

The Photosynthetic Membrane

In our discussion of photosynthesis have we thus far only regarded biochemical reactions. In the section about glycolysis and other biosynthetic pathways was the significance of single enzymes pointed out. Since quite some time know, for example, have all enzymes involved in glycolysis been purified and isolated and each step of the pathway can be analyzed under in vitro conditions. It has also been tried to isolate the complete set of components necessary for photosynthesis in order to reconstitute the whole system. But all attempts failed because the premises they were based on were wrong as we know today.

A number of problems have not been taken into consideration until now. The terms photosystem I and photosystem II, for example, have been introduced and all participating pigments have been mentioned but the following subjects remain to be discussed:

How are the photosystems organized?How are the pigments arranged?Why does one of the chlorophyll molecules react different than all the others?Why are action and absorption spectra not quite congruent?Why reacts P 680 (chlorophyll a) different than P 700 (chlorophyll a, too)?How are electron transport chain and ATP production coupled?How are photosystem I and II linked?Which structural prerequisites have to exist in order for the two systems to co-operate?

It was always accepted that each of the biochemical reactions was catalyzed by a specific enzyme and still, it took quite some time before it was realized that the chlorophyll and the other pigments are protein-bound and that they are only active as protein-chlorophyll (and protein-pigment, respectively) complexes. The isolated pigments themselves were useless for photosynthesis. The pigment-protein complex, (most) proteins of the electron transport chain as well as the catalyst of ATP synthesis (ATP synthase) are integral compounds of the photosynthesis membrane(s) (= the thylacoid membranes of algae and higher green plants, cytoplasmatic membranes of photosynthetically active bacteria and blue-green algae). The location within the membrane (at the out- or the inside, for example) and the relative arrangement of the proteins towards each other are important prerequisites of energy transformation.

This is not only true for photosynthetic reactions but also for those of the respiratory chain and for the enzymes located within the purple membrane of Halobacterium halobium (an archaebacterium using light energy for the production of ATP without an electron flow).

The requirements for energy transformation are even higher: completely intact membranes that are impermeable for protons and that enclose compartments thus maintaining a stable electrochemical gradient between inside and outside. The production

of ATP is based on a directed proton dislocation paralleled by a change of the compartment's pH and of its membrane potential.

Proteins of The Photosynthetic Membrane

The research into the proteins essential for photosynthesis started very late. The reason is that all of them are membrane-bound which rendered it nearly impossible to isolate and characterize them with the classical methods of protein analysis.

Only after sensitive techniques like gel electrophoresis and the controlled use of detergents like sodium dodecyl sulfate (SDS) had been developed, became it possible to separate the proteins and to identify them as bands in a gel. A side product of this technique is the determination of the molecular weights of the respective polypeptide chain.

A second, independent attempt was and is the use of specific probes like fluorescence-tagged antibodies that help to find out whether a certain protein (or part of a polypeptide chain) is located at the inside or the outside of a membrane. The use of antibodies against specific proteins allows, too, to precipitate these proteins selectively since only they are able to form the extremely specific antigen - antibody complex.

Cross-linking agents render it possible to elucidate the surrounding of a molecule. And the use of specific inhibitors helps localizing their site of effect. DCMU [3-(3', 4' - dichlorphenyl) - 1,1 - dimethylurea] has since years been used to inhibit photosystem II. It has no effect on photosystem I and was therefore used by ARNON and his collaborators as an important help to study the electron transport chain that starts at photosystem I independently of that induced by photosystem II.

We know today that DCMU does not effect chlorophyll itself but a certain protein, the plastoquinone-binding protein.

A third possibility to characterize the photosynthetic membrane is the analysis of certain mutants. The single-celled alga Chlamydomonas reinhardii proved to be a good test object. Quite a range of mutants with photosynthetic defects are known. They can be grouped in four classes:

1. mutants with a defect in photosystem I, 2. mutants with a defect in photosystem II, 3. mutants with a defect in photophosphorylation and 4. mutants with a defect in the antenna complex.

It is quite striking that almost all mutants are characterized not only by the loss or change of a certain polypeptide chain but by the lack of a whole complex, for example that of PS I. It seems therefore as if the mutations would lead to pleiotropic effects. Or, expressed

differently: when a polypeptide chain is changed or missing does the assembly of the other polypeptide chains not work any more. This observation shows how tight the interactions between the single polypeptide chains are and how important they are for their mutual co-operation.

A further and not less important technique is electron microscopy usually used in combination with freeze-etching.

The sequencing of membrane proteins remains difficult. And yet, the sequences of most proteins involved in photosynthesis could be determined during the last years via the sequencing of their respective genes. The most remarkable outcome of this work is that these proteins contain (just like proteins of animal or bacterial membranes, too) a large portion of alpha - helices. The lengths of the helices corresponds to the thickness of the membranes. The single helices are connected via polar and / or non-hydrophobic sequences.

Chlorophyll - Binding Proteins

Several chlorophyll-binding proteins of the photosynthetic membranes of different systematic groups (angiosperms, gymnosperms, algae, bacteria) have been isolated and characterized. The best-known are P 700-chlorophyll-a-protein 1 and the light-harvesting chlorophyll-a/b-protein 2 which have been studied in the laboratory of J. P. THORNBERGER at the University of California, Los Angeles. Both are strongly hydrophobic, integral membrane proteins. Both bind chlorophyll a but only the latter binds chlorophyll b, too. The P 700-chlorophyll-a-protein 1 contains the reaction centre (P 700) of photosystem I, i.e. one of the chlorophyll molecules is bound in a specific configuration and is located in a surrounding (due to a specific amino acid composition and the folding of the polypeptide chain) that renders it different than all other chlorophyll molecules bound by this protein, too. This structural peculiarity is the precondition for the light-induced activation and consequently for the induction of the electron flow.

The molecular weight of the polypeptide chain is 110,000 Dalton. It is able to bind 14 chlorophyll molecules. The light-harvesting-protein (light-harvesting chlorophyll-a/b-protein 2) is also very common. It is mainly associated with photosystem II but effects on photosystem I have been observed, too. Chlorophyll a and b are bound in equimolar amounts besides lutein and beta - carotene. The chlorophyll to carotenoid ratio is 3 - 7 : 1 on a molecular level.

Structure of the light harvesting complex of photosystem II - Arrangement of Pigments

© Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign, a-crofts@uiuc.edu

Light Harvesting Complexes I and II (LH I, LH II) and Reaction Centre (RC)

©: Theoretical Biology Group - University of Illinois at Urbana-Champaign

The Coupling Factor: an ATP - Synthase

ATP - synthase is the enzyme that catalyzes the synthesis of ATP. Since the production of ATP occurs not only during photosynthesis but during respiration, too, suggested the idea that the ATP production of both cases is based on similar mechanisms itself.

After having collected experience with the mitochondrial ATP synthase, did E. RACKER of the Cornell University isolate an enzyme of thylacoid membranes that resembled the respective mitochondrial enzyme very much. In the electron microscope did it look like a stemmed knob. The knob was termed CF1 and the stem CF0 (F1 and F0 respectively in the mitochondrial enzyme). CF0 (or F0) is a membrane anchor. While the F1 - F0 complex is localized in the inner mitochondrial membrane and the knob is directed towards the mitochondrial matrix are the respective molecular parts of the CF0 - CF1 complex found at the outside of the thylacoid membranes. In both cases consists the ATP synthase out of several different polypeptide chains, it is an enzyme complex. The phosphorylation of ADP works only, if the ATP synthase is a component of an intact, proton-permeable membrane. It has to separate two compartments (the inner part of the vesicle and the surrounding).

©Copyright 1996, Antony Crofts, University of Illinois at Urbana-Champaign, a-crofts@uiuc.edu

The Chemiosmotic Hypothesis of P. MITCHELL: a Model of The Photosynthetic Membrane

Since quite some time has the ATP synthase been ascribed the function of a coupling factor. This means that it is able to utilize the free energy released by electron transport. Such energy conservation is referred to as energy coupling or energy transduction.

How does this work? It might have been assumed that the electron transport chain serves the production of energy-rich intermediates and that these constitute an energy store for the production of ATP. Two arguments against this idea exist:

1. No such substances have ever been isolated and

2. photophosphorylation (and the oxidative phosphorylation of the respiratory chain, respectively) is only possible if the thylacoid membranes (the inner mitochondrial membrane, respectively) are intact.

Another assumption had been that the ATP synthase changes its configuration and is thus itself transferred into an activated state. In such a case would the energy be transiently stored in weak interactions. This hypothesis, too, failed to withstand experimental scrutiny.

In 1961 proposed P. MITCHELL (Glynn Research Laboratories, Great Britain) that the energy set free during the electron transport is conserved as a proton gradient across the membrane. The energy would then not be stored as a chemical bond but as an electrochemical gradient. The electrochemical potential of this gradient would be harnessed to synthesize ATP. The hypothesis explains several key observations:

1. It is consistent with the fact that oxidative phosphorylation requires an intact inner mitochondrial membrane.

2. The inner membrane is impermeable to ions like H+ or OH- whose free diffusion would discharge the electrochemical gradient.

3. The electron transport results in the transport out of intact mitochondria (out of the thylacoid space of chloroplasts) thereby creating a measurable electrochemical gradient across the inner mitochondrial membrane (the thylacoid membrane of chloroplasts).

4. Substances that increase the permeability of the inner mitochondrial or the thylacoid membrane to protons, thereby dissipating the electrochemical gradient, allow electron transport to continue but inhibit ATP synthesis: they 'uncouple' electron transport from oxidative phosphorylation.

A very convincing experiment was performed by A. T. JAGENDORF (1966, Cornell University, Ithaca, N. Y.): he took isolated thylacoids and incubated them in a pH buffer until the same pH (4) was measured at both sides of the membrane. After the equilibrium had been achieved, did he quickly transfer the thylacoids to a media of pH 8 containing both ADP and Pi. Immediately after the transfer was the pH between inside and outside evened out while, at the same time, the system produced ATP. The proton flow across the membrane was used for the production of ATP. The experiment worked only with intact membranes. In addition have all biochemical processes to be directed (vectorial). All enzyme molecules have to have the same direction so that the protons are transported in only one direction.

How can a flow of protons induce the production of ATP??

To understand the process is it useful to study the ATP production a little more. ADP and phosphate (Pi) are its starting compounds. It is known that both are bound to neighbouring but separate binding sites of the

enzyme complex (the ATP synthase ). To produce ATP (ADP~P) has one H+ to be removed from ADP and one OH- from phosphate. In a formal sense is water split off. As soon as the ions have left the complex do both combine with their counterions to water (not with each other). The end result is a directed flow of protons.

This does not mean that one proton is transferred through the membrane via the ATP synthase complex. Instead is a newly formed proton given off into solution at one side while another proton is captured and neutralized (by a OH- ion) at the other side of the membrane.

Independent of these observations could GRÄBER and WITT (Max-Vollmer-Institut, Technische Universität Berlin) show that a direct coupling between proton gradient and the electron transport chains of the photosystems I and II exists. It emerged that the already known 'Z'-scheme is no hypothetical product but that the involved components are arranged like a Z within the photosynthetic membrane, i.e. the structural basis for the process discussed in the previous sections became known.

D. von WETTSTEIN and R. P. OLIVER (Carlsberg Laboratorium, Copenhagen, 1985) summarized all results and were thus able to develop a model that explains the topology of the single protein complexes.

The photosynthetic membrane contains four complexes altogether. Each has subunits encoded in the nucleus and others encoded by plastids. Several proteins bind chlorophyll a, one binds chlorophyll a and b. The PS II complex is mainly localized in stacked, PS I and the ATP synthase complex (CF1 - CF0) in non-stacked thylacoid membranes.

A further remark: the reactions of the CALVIN cycle are catalyzed by soluble enzymes, localized within the stroma.

Molecular Structure of The Reaction Centre

In 1985 was the structure of the photosynthetic reaction centre's protein subunits determined. J. DEISENHOFER, H. MICHEL and R. HUBER (Max-Planck-Institut für Biochemie, Martinsried) succeeded in crystallizing the protein complex of the photosynthetic membrane of the bacterium

Rhodopseudomonas viridis, they determined the folding of the polypeptide chain and the arrangement of the chlorophyll molecules. In 1988 were they awarded the Nobel prize for this work. Together with the tertiary and quaternary structure was also the amino acid sequence of the involved polypeptides determined. The central part of the complex contains two subunits, L and M, each of which forms 5 helices that span the photosynthetic membrane. Two further polypeptides, H and a cytochrome c - like protein are associated. Furthermore belong 4 covalently linked heme groups, 4 bacteriochlorophyll b molecules, 2 molecules of bacteriopheophytin b, 2 quinones, 1 iron ion that is not linked to a heme group as well as carotenoids as prosthetic groups to the complex. The structures found in bacteria are homologous to the reaction centres of the photosystems I and II of green plants.