Photosynthesis

The Dark Reactions of

Photosynthesis, Assimilation of
Carbon Dioxide And The
Calvin Cycle
C3, 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: Grundzge 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] fr

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 OHThis 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 H218O, 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 (twodimensional 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 + C 1 = 2 C 3
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
o

the resynthesis of ribulose diphosphate and

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:

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.

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-7phosphate (S-7-P) with glycerinaldehydephosphate. Two C5 units are the result:
ribulose-5-phosphate (Ru-5-P) and xylulose-5-phosphate (X-5-P).
o

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 3phosphoglycerate 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 HATCHSLACK-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.
BJRKMAN 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. LSCH and H. KAPPEN, Universitt 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 lightinduced 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 noncyclic 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 fluorescencetagged 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.
2.
3.
4.

mutants with a defect in photosystem I,

mutants with a defect in photosystem II,
mutants with a defect in photophosphorylation and
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/bprotein 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.
2.
3.
4.

It is consistent with the fact that oxidative phosphorylation requires an intact inner mitochondrial
membrane.
The inner membrane is impermeable to ions like H+ or OH- whose free diffusion would discharge
the electrochemical gradient.
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).
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 (P i) 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 GRBER and WITT (Max-Vollmer-Institut, Technische
Universitt 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 (CF 1 - CF0) in nonstacked 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 fr 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.