Download intermediary metabolism

1
INTERMEDIARY METABOLISM
Photosynthesis
Dr. (Mrs.) Veena Taneja
Professor of Biochemistry (Superannuated)
Banaras Hindu University
Varanasi - 221 005
0 2 March 2007
CONTENTS
Chloroplast
Thylakoid membrane
Thylakoid lipids
Thylakoid proteins
Overview of Photosynthsis
Light Reactions
Hill Reaction
Major molecular complexes of light reaction
Absorption of sunlight
Photosynthesis in higher Plants, alage and cyanobacteria
Inhibitors of light reactions
Bacterial photosynthesis
Why two photosystem in Higher Plants, Algae and Cyanobacteria?
Dark Reactions
Calvin Cycle
Efficiency of photosynthesis
Photo respiration
C4 Pathway
Crassulacean acid metabolism (CAM)
Keywords
P h o to s yn thes i s, Ch lor o p l as t,
L ig h t Re a c t io n s , P h o to s yst e m I ,
P h o to s y s t e m I I , L ig h t
H arvesting Co mp lex, ATPSyn th ase, Pho tophosphor ylation, D ark Reaction s,
Calv in Cycle,
Rub isco, C3- and C4- Pathways, CA M
2
Photosynthesis is the process where by many plants, algae and microorganisms synthesize
carbohydrates from carbon dioxide and water using the energy of the sun:
light
6CO2 + 6H2O ⎯⎯
⎯→ C6H12O6 + 6O2
(i)
Photosynthesis is important because the carbohydrates synthesized are utilized not only by plants
themselves but also by other living organisms (Herbivores) directly (by consuming the plants) or
indirectly by animals (carnivores) who eat other animals dependent on plants and it releases O2
into the atmosphere. Most living organisms consume O2 to oxidize carbohydrates and other
substrates to produce ATP for their energy and other metabolic demands such as a source of
building units to produce other biomolecules - (amino acids, proteins and fats etc) and release
CO2 into the atmosphere. Thus, Photosynthesis maintains the carbondioxide (CO2) and oxygen
(O2) levels in the atmosphere. Almost half of the CO2 is fixed by algae in the ocean and the rest,
mostly by tropical rain forest.
Chloroplast
The mesophyll cells of the leaves of plants contain chloroplasts which are the organelles of
photosynthesis. Carbon dioxide enters and O2 exits the leaf through pores called stomata
(singular stoma, mouth). Water is absorbed by roots and reaches the leaves through veins, which
also carry the synthesized carbohydrates to other parts of the plant. Carbondioxide enters the
chloroplast membrane as such and not as H2CO3 or HCO-3.
Each mesophyll cell of plants may contain 20 to 50 chloroplasts, sometimes even 100 or more
depending on the species, cell type and growth conditions. Algae cells, also have chloroplasts but
often each cell has one large chloroplast. Some prokaryotes, which carry out photosynthesis, do
not contain chloroplasts but their membrane structures play the same role as do the chloroplast
membrane system.
Chloroplasts are watermelon shaped and measure 2 to 4µm by 4 to 7µm. They are very similar
to mitochondria. They are autonomous, have their own Deoxyribonucleic acid (DNA) and
protein synthesizing components i.e. ribosomes, Ribonucleic acids (RNA) and relevant enzymes.
They also contain proteins encoded by nuclear genes and an intricate membrane system. The
chloroplast has three different membranes viz the outer, inner and thylakoid and three separate
spaces viz the inter membrane space, stroma and the thylakoid space or the lumen of the
thylakoid (Figure 1).
The outer membrane is freely permeable. The inner membrane, which is semi- permeable, has
transport proteins. The intermembrane space is between the outer and the inner membrane within
the chloroplast. The inner membrane encloses a dense fluid, the stroma, comparable to
mitochondrial matrix. In the stroma, there is an interconnected membrane system, the thylakoid
membranes. These are flat sac like structures which are piled one on top of the other to form
discs called 'grana' (singular granum) or stacked or appressed thylakoid, These grana are
connected irregularly, by stromal lamellae or unstacked or unappressed thylakoid. The thylakoid
membranes enclose a single continuous space known as lumen of the thylakoid. A chloroplast
contains about 10 to 100 grana. The grana increase the surface area of the thylakoid membrane
as do the 'cristae' of inner membrane of the mitochondria (Figure 1).
3
In developing chloroplasts, the thylakoid arise from invaginations of the inner membrane
analogous to the mitochondrial cristae.
Figure 1: Structure of Chloroplast (on the left is the electron micrograph of a chloroplast
showing the thylakoid membranes stacked into grana in the stroma. The membrane systems of
the chloroplast discussed in the text are shown in the figure on right)
(Source: Campbell, N.A., Reece, I.B. and Mitchell, L.G., 1999. Biology. 5th edition.
Benjamin/Cummings Publishing company)
Thylakoid membranes
The thylakoid membrane like all membranes, consists of a lipid bilayer and proteins. The lipid
and protein are in the ratio of nearly 1:1. The thylakoid membrane exhibits a high degree of
fluidity and allows free lateral movement of the components within the plane of the membrane.
The thylakoid also exhibit asymmetry and crossing of the molecules from one half of the
membrane to the other half is restricted.
Thylakoid lipid
The lipid composition of these membranes is very characteristic. Of the total lipids, glycolipids
are 40%, sulfolipids 4%, phospholipid 10% and the photosynthetic pigments 20%.
The major glycolipids, monogalactosyl- and digalactosyl-diglycerides, sulfoquinovosyl
diglyceride (also called plant sulfolipid [Figure 2(a)] and the phospholipids are the structural
lipids. The R1 and R2 of the chloroplast lipids is the unsaturated fatty acid-α linolenic acid which
is the major component (90% in some cases) while trans-3-hexa decenoic acid is the minor
component [Figure 2(b)].
The photosynthetic pigments comprise of chlorophyll a, chlorophyll b, carotenes and
xanthophylls in plants. In cyanobacteria (blue green algae) red algae, the pigment are the blue
cyanobilin and red phycoerythrobilin, while in photosynthetic bacteria the pigments are bacterio
4
chlorophyll a and b (Figure 3). The electron dense tetrapyrrole of the chlorophyll molecules is
responsible for the light absorption and the long hydrophobic phytol tails help to anchor the
molecule in the thylakoid membrane.
(a)
(b)
Figure 2 (a) Structure of the chloroplast galactolipids and of sulfoquinovosyl diglyceride
where R1 and R2 are fatty acids; (b) Fatty acids found in chloroplast lipids
Proteins
The protein components of the thylakoid membrane in plants are about 30-50 polypetides which
are present in five major supramolecular complexes, which have been isolated by treatment with
mild detergents. They are:
1. Photo system I (PS I)
2. Photo system II (PS II)
3 Light Harvesting Complex
(LHC)
4. Cytochrome b6f (Cyt b6f)
5. ATP Synthase (CF0-CF1, coupling factor)
PSI, PSII and LHC are chlorophyll protein complexes where all the chlorophyll is associated
with the proteins noncovalently in the thylakoid membrane.
5
Figure 3: Structure of photosynthetic pigments – (a) Chlorphylls a and b and bacterio
chlorophyll; (b) Phycoerythrobilin and phycocyanobilin, the antenna pigments in
cyanobacteria and red algae; (c) β-carotene and (d) Lutein (xanthophylls)
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
Overview of Photosynthesis
The equation (i) given for photosynthesis is an over simplification. It actually involves many
intermediate steps.
Biosynthetic processes require an energy source and a reductive power, NADPH. Thus, the
energy of the sunlight is converted into the energy currency of the cell i.e. ATP and reductive
6
power NADPH in a series of reactions occurring in the thylakoid membranes known as LIGHT
REACTIONS.
+
+
light
12H2O + 12 NADP+ + 18ADP + 18P2 ⎯⎯
⎯→ 12 NADPH 12H 18 ATP + 6O2
The two products of the light reactions, ATP and NADPH then reduce CO2 in a series of
enzymatic reactions which occur in the stroma, called the DARK REACTIONS (so called as
they are light independent). However some of the reactions occur optimally in presence of light.
The two set of reactions i.e. the light and the dark reactions are physically and chemically
separate.
The principal end products of photosynthesis are sucrose, starch and oxygen. Starch is stored in
the chloroplast while sucrose is transported from the leaf to other parts of the plant. Oxygen is
released into the atmosphere via stomata.
Light reactions
In higher plants, algae and cyanobacteria H2O is the ultimate reducing agent in photosynthesis.
However, some photosynthetic bacteria use other reductants such as HSO3-, H2, lactate etc.
The general reaction is given by
CO2 + 2H2A ⎯⎯→ [CH2O] + H2O + 2A
where, H2A is a general reductant and A is the oxidized product.
H2A is H2O in green plants and cyanobacteria and H2S in photosynthetic sulfur bacteria etc.
(Table 1)
Organisms
Plan ts , algae, cyanobacter ia
R educta nt
H2O
R eact io n
G r een su lfu r b a c te r ia
H2S
CO 2 + 2H 2 S ⎯⎯→ [ CH 2 O ] + H 2 O + 2 S
P u r p l e su lfu r b a c te r ia
[HSO 3− ]
CO 2 + H 2 O + 2 [HSO 3− ] ⎯⎯→ [ CH 2 O ] + 2 [HSO −4 ]
Non su lfur pho tos yn th e tic
b ac ter ia
H 2 or ma n y o th er
r educ tan ts su ch
a s l a c ta t e
CO 2 + 2H 2 ⎯⎯→ [CH 2 O ] + H 2 O
CO 2 + 2H 2 O ⎯⎯→ [ CH 2 O ] + H 2 O + O 2
CH3
CH3
CO2 + 2 HC — OH ⎯⎯→ [ CH 2 O ] + H 2 O + 2 C = O
COO −
COO −
Lactate
Pyruvate
Table 1: Example of some photosynthetic reactions
(Source : Campbell, N.A., Reece, I.B. and Mitchell, L.G., 1999. Biology.5 t h edition.
Benjamin/Cummings Publishing company)
7
In early 1930s, C.B.Van Niel predicted that during photosynthesis O2 is evolved from H2O and
not from CO2. In 1941, labeling experiments confirmed this. It was shown using H218O and
unlabelled CO2 that neither of the oxygen atoms come from CO2 and that one of the oxygen from
CO2 ends up in carbohydrate.
18
light
CO2 + 2H218O ⎯⎯
⎯→ [CH2O] + H2O + O2
Hill reaction
Many scientists working in different laboratories over the years contributed to the understanding
of the photochemical light reactions.
In 1939, Robert Hill showed that when isolated chloroplasts were illuminated in the presence of
any of a variety of acceptors, they promote reduction.
2+
+
light energy
4Fe3+ + 2H2O ⎯⎯
⎯ ⎯⎯→ 4Fe + 4H + O2
A number of such reactions using different inorganic oxidants are known and are referred
to as HILL REACTION. These reactions in the absence of photochemical activation are
unfavourable. Hill reaction showed that chloroplasts when irradiated by sunlight could oxidize
water to O2 in the absence of CO2. Thus, for the first time it was shown that the light and dark
reactions are separate. It was ultimately found that
(1) the final acceptor of the light reactions in vivo is NADP+ in higher plants and
(2) light reactions generate a proton gradient which drives the ATP synthesis by CF0-CF1,
ATPase..
Major molecular complexes of light reactions
The major molecular complexes involved in the light reactions have preferential localization in
the thylakoid membrane (Figure 4) and are discussed below:
Photosystem I and Photosystem II
The light harvesting complex, electron carrying cofactors, proteins and the reaction centre are
packed into supramolecular complexes as a unit called Photosystem complex. Associated with
each Photosystem (PS) complex are about 200 to 400 molecules of pigments which act as
'LIGHT TRAPPING ANTENNAE'. It also contains a special pair of chlorophyll a which is the
Reaction Center Chlorophyll. The antenna chlorophyll is different from the reaction center
chlorophyll.
The photosynthetic bacteria contain bacteriochlorophyll and have only one photosystem while
plants, algae and cyanobacteria have two photosystems, Photosystem I (PSI) and Photosystem II
(PSII).
8
Figure 4: Localization of the major complexes, PSI, PSII, Light harvesting complex II,
Cytochrome b6f complex and ATP synthase in the thylakoid membrane
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
The Photosystem with P700 was discovered first and hence named, PSI, Here the reactive
chlorophyll a dimer is P700 where P stands for pigment and 700 is the wavelength at which it
absorbs light. The photosystem with P680 was discovered later, hence named PSII. The reactive
chlorophyll a dimer is P680 and 680 is the wavelength at which it absorbs light. PSI has a high
ratio of chl a/chl b, whereas in PS II the ratio of chl a/ chl b is nearly one.
PSI and PSII in plants are spatially segregated and are 100Å apart where PSI is in the unstacked
and PSII in the stacked thylakoid membrane.
Photosystem I
PSI is related to that of green sulfur bacteria. It is about 800 kd transmembrane complex. P700
traps the energy quanta excited by the absorption of light by Antenna chlorophyll. Its unusual
properties are due to its association with specific proteins in the thylakoid membrane. The core
PSI consists of thirteen polypetides.
(1)
(2)
(3)
(4)
(5)
Two large nearly identical proteins, PsaA and PsaB (83 and 82 kd respectively) form a
dimer which contains P700; Ao-a special chlorophyll, A1, a-quinone (vitamin K1) and Fx,
FA and FB, the three Fe-S proteins. A0, A1 and Fx, FA and FB are the electron
acceptors.
A small protein, Psa C (9 kd) which contains Fe-S centers.
Extrinsic protein Psa F binds soluble plastocyanin, a mobile electron carrier, to the
lumenal side of PSI.
Psa D, an extrinsic protein, binds ferridoxin to the stromal side of PSI.
Small peripheral proteins, PsaE, PsaI to M and few others.
9
PSI accepts electron from plastocyanin and reduces ferridoxin (a Fe-S protein) soluble in stroma,
which ultimately reduces NADP+ to from NADPH.
PSI of cyanobacteria has been studied in detail and its crystal structure worked out. It consists of
11 subunits Psa A to F and Psa I to M. All protein components except (Psa C) have long αhelices spanning the membrane.PsaA/B(83 kd) transmembrane dimer in the thylakoid contains
reaction center.
Photosystem II
PSII is similar to the photosystem reaction center of purple bacteria which do not from oxygen
and both appear to have arisen from a common ancestor.
PSII is about 600kd, transmembrane assembly of more than 10 polypeptides. The core PSII
consists of:
(1)
(2)
Two core, integral proteins D1 and D2 (each 32 Kd) They bind two P680 reaction center
special pair of chlorophyll, two other chlorophylls, two pheophytins, one Fe atom and
two quinones (QA and QB) which are the electron carriers.
Three extrinsic proteins (33, 23 and 17 kd respectively) which form the Oxygen Evolving
Complex (OEC) or the water splitting enzyme, present on the lumenal side of the
thylakoid membrane. OEC binds four Mn2+, Ca2+, Cl-1 that function in the splitting of
water and maintain the essential environment.
PSII transfers electrons from water to plastoquinone pool.
Light harvesting complex (LHC)
Light harvesting complex (LHC) consist of one or more polypeptide chains containing
photosynthetic pigments which surrounds a photosynthetic reaction center in the photosystem to
form photosystem complex (Figure 5). LHC can absorb visible and near infrared light and
contains half of the total chlorophyll. Its function is to capture solar energy and focuses light
inwards towards its core. Most of the chlorophyll molecules are not directly involved in the
photochemical process itself. They function to gather light and act as antenna molecules, Most of
the chlorophyll is associated with proteins which may provide the correct orientation of the
chlorophyll molecule for efficient transfer of absorbed light energy between them. The energy
absorbed by carotenes can be directly transferred to the chlorophyll molecules.
Light harvesting complex associated with PSI is termed LHC-I and that with PSII LHC-II. Also,
there is a mobile pool of LHC. The two LHCI and LHC II have differences in their polypeptide
chains. Most of the LHC surrounds PS II. LHC I binds approximately 9 chl molecules but the
total number and composition of carotenoids associated with LHC-I is variable. LHC-II is the
most abundant membrane protein in plant chlorophasts. A major LHC-II protein binds 7 chl a
molecules and 6 chl b molecules and 2 carotenoid molecules and is called chl a/b LHC. It
appears that LHC in plants consist of membrane bound hydrophobic proteins having membrane
spanning
α - helices and each containing several pigment molecules. LHCII from pea
chloroplast has been studied by electron crystallography [Figure 5(b)].
10
a
b
c
d
Figure 5: (a) Structure of a light harvesting complex. Eight polypeptides, each of which
binds three chlorophyll molecules and a carotenoid molecule, surround a
central cavity that contains the reaction centre.
(Source : Berg, J.M., Tymoczko, J.L. and Stryer, L. 2002. Biochemistry. 5 t h
Edition W.H. freeman and company, New York)
(b) Three dimensional structure of a monomer of the trimeric, Light harvesting
complex of plants. The transmembrane protein has membrane spanning
helices and seven chlorophyll a, five chlorophyll b and two carotenoid
molecules.
(Source: Campbell, N.A., Reece, I.B. and Mitchell, L.G., 1999. Biology.
5th edition. Benjamin/Cummings Publishing company)
(c) The x-ray structure of a subunit of the homotrimeric bacteriochlorophyll a
protein from P. aestuarii, the polypeptide backbone of one subunit consists
largely of a 15-stranded antiparallel β sheet with seven bound bacterio
chlorophyll a molecules with their Mg2+ ions shown in spheres.
(Source:Voet, D and Voet J. 1995. Biochemistry. John Wiley & Sons, USA)
11
(d) Structure of a phycobilisome. Schematic representation of a phycobilisome
from the cyanobacterium synechocystis 6701. Rods containing phycoerythrin
(PE) and phycocyanin (PC) emerge from a core made of allophycocyanin
(AP) and allophycocyanin B (APB). The core region binds to the thylakoid
membrane.
(Source : Berg, J.M., Tymoczko, J.L. and Stryer, L. 2002. Biochemistry. 5 t h
Edition W.H. freeman and company, New York)
X-ray structure of an antenna protein from green photosynthetic bacterium, Prosthecochloris
aestuarii, has been determined by Brian Matthews. It is a trimeric protein. Each subunit has 7
bacteriochlorophyll a molecules and consists of 15 strands of antiparallel β-sheet wrapped
around the chlorophyll containing core. It appears as a string bag holding the pigment molecules
[Figure 5(c)].
Phycobilisomes
In cyanobacteria (blue green algae) and red algae, the antenna pigment, Chl a is replaced by bilin
(tetra pyrroles)-mostly the blue phycocyanobilin and the red phocoerythrobilin. These bilins are
covalently attached to cysteine groups of phycobili proteins which form large (several million
dalton) assemblies called phycobilisomes which are bound to the outer face of the thylakoid
mambrane. These antenna pigments absorb the green and the yellow light (in the range of 400650nm) which reaches them and funnel excitation energy within picoseconds to PSII reaction
centers with >90% efficiency. These light pipes [Figure 5(d)] help the algae to
survive in their natural habitat-sea water at depth of a meter or so where little blue or green light
reaches them.
Cytochrome b6f Complex (C/tb6f Complex)
It is a membrane spanning complex consisting of 4 subunits:
(1)
(2)
(3)
(4)
Cytochrome f (cyt f), (where f is frons, French for leaf) which is a 33 kd subunit.
Cytochrome b563 with two hemes, is a 23kd subunit.
a 20 kd (Fe-S) protein with (4Fe-4S) centers and
a 17 kd polypeptide subunit.
Cyt b6f complex is uniformly distributed in the thylakoid membrane.
It accepts electron from plastoquinone (PQ), which diffuses in the membrane. Cyt b6f donates
electron to the mobile one electron carrier protein, plastocyanin (PC).
ATP Synthase (CF0-CF1, AT Pase)
The plant ATP synthase (synthetase) is also known as CF0-CF1 ATPase (where C stands for
chloroplast). It is a transmembrane multisubunit enzyme complex located in the unstacked region
of the thylakoid membrane.
The structure and mechanism of ATP synthesis by CF0-CF1 ATPase is analogous to that of F0-F1
ATPase of mitochondria and that of bacteria. It appears like a "lollipop" or a stemmed knob. The
12
knob or the head piece is the CF1 which is a hydrophilic peripheral protein. It is attached to the
base piece, CF0, which is a hydrophobic transmembrane anchor.
CF0 consists of 4 types of subunits, I, II, III and IV whose stoichiometry is estimated to be
1:2:12:1. The subunit III is a proton translocating channel which has certain strategically located
carboxyl (COO-) groups of Aspartate / glutamate lining the channel. The binding of dicyclohexyl
carbodiimide, DCCD, to these COO- groups inhibits the transport of protons through CF0-CF1
complex and consequently ATP synthesis. The proton flow through CF0 is from the lumen of the
thylakoid to the stroma.
CF1 consists of five subunits α3β3γδε The α and β subunits are arranged alternately in CF1 and
the β subunit has the enzyme active site for ATP synthesis. The δ subunit binds CF1 to CF0 while
the γ subunit forms the gate to control the proton flow from CF0 to CF1. The ε subunit inhibits
the catalytic activity in the dark. The proton flow drives the release of the ATP synthesized by
the ATP synthase according to the equation:
ADP + Pi ⎯⎯→ ATP + H2O
All ATP synthases whether from chloroplast, mitochondria or bacteria are rotary motors in
which energy is derived by passage of protons and are reversible enzyme complexes that can
hydrolyse ATP if the proton gradient is reversed.
Absorption of sunlight during photosynthesis
Photosynthesis depends on light absorbed by photosynthetic pigments (Figure 6) in the visible
and near infrared region of the spectrum. Chlorophyll a is the main pigment which absorbs light
in the blue (420-435nm) and red (660 to 680nm) part of the spectrum. Chlorophyll b absorbs
blue green light in the region 400-500 nm more efficiently than chlorophyll a (Figure 6). The
chlorophyll a and chlorophyll b complement each other in absorbing incident sunlight.
Carotenoids also absorb light efficiently in this region i.e. 400-500 nm. They also serve a
protective role, by suppressing the damaging photochemical reactions involving oxygen which
can occur on exposure to sunlight. Chlorophyll b, carotenoids and xanthophylls are called
accessory pigments and are important in LHC of higher plants. Most of the solar energy that
reaches the earth's surface lies in this range.
Light can be thought of as discrete packets of energy (quanta) called photons. The energy of the
absorbed photons is dependent in the wavelength at which light is absorbed. Since the
chlorophyll and other pigments and proteins are closely packed in the photosystem, when the
chlorophyll molecules absorb the light energy, the electrons in the π-orbital of the conjugated
bond system are excited from the ground state to a higher electronic state. This excitation energy
is passed from one molecule to the adjacent molecule by resonance transfer or the excited
electron itself may pass to a nearby molecule at a lower excited state by electron transfer
reaction. Both these processes are important in photosynthesis. (Figure 7). Exciton is the
quantum of energy passed from an excited chlorophyll molecule to the adjacent molecule in a
process called exciton transfer by inductive resonance.
13
Figure. 6: Absorption of visible light by photosynthetic pigments. The spectrum of sunlight
reaching the earth’s surface is also shown.
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
Figure 7: Resonance transfer of energy in a Light harvesting complex
(Source: Campbell, N.A., Reece, I.B. and Mitchell, L.G., 1999. Biology.
5th edition. Benjamin/Cummings Publishing company)
The antenna chlorophyll a pigment molecule of light harvesting complex, absorbs photons and
the energy is passed by exciton transfer randomly to adjacent chlorophyll molecules till it is
trapped by a special pair of chlorophyll a in the specific reaction center. Only the special pair of
14
chlorophyll a molecule within the reaction center can participate directly in the conversion of
photochemical energy to electrochemical energy. This then starts a series of electron transfers.
Photosynthesis in higher plants, algae and cyanobacteria
The two photosystems, PSII and PSI, are required for maximal efficiency of photosynthesis in
higher plants, algae and cyanobacteria. They are linked in series to carry out the sequence of the
light reactions depicted in Figure 8.
Figure 8: Light reactions of photosynthesis in plants, which utilize two photosystems, PSI
and PSII, showing the reduction of NADP+ and ATP synthesis
(Source : Lodish, H., Baltimore, D., Berk, A., Zipursky, S.L., Metasudaira, P. and
Darnell, J. 1995. Molecular cell Biology, 3 r d Edition, Scientific American Books W.H.
freeman and company, USA)
Since the direction of spontaneous electron flow is from a more negative reduction potential to a
more positive reduction potential, the spontaneous electron flow from PSII to PSI and that from
ground state of P700 to NADP+ is not possible, as the reduction potential of P680 in PSII is more
positive than that of P700 in PSI. Also, the reduction potential of P700 is more positive than that
of NADP+. These thermodynamically unfavourable reactions are possible as a result of the
energy provided by the sunlight.
15
Z scheme or the linear electron flow by the two photosystem light reactions
The pathway of electrons from H2O to NADP+ through the major electron carriers of the two
photosystems, PSII and PSI when graphed on a scale of reduction potential results in a zigzag
pattern referred to as 'Z' scheme (Figure 9)
Photosystem I
P700*
A0
Photosystem I
A1
–1.0
P680*
Fe-S
Pheo
Fd
PQA
Fd:NADP+
oxidoreductase
E’0 (volts)
PQB
Light
Cyt
b6 f
complex
0
NADP+
NADP+
Plastocyanin
Light
Proton
gradient
H2O
1.0 1/2O
2
O2
evolving
complex
e−
P680*
P700*
PQA = plastoquinone
PQB = second quinone
A0 = electron acceptor chlorophyll
A1 = phylloquinone
Figure 9: The ‘Z’ scheme showing the pathway of non cyclic electron transfer from H2O to
NADPH. The dashed arrow from ferridoxin to cytochrome b6f complex is the path of cyclic
electron transfer.
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
Electron transport through photosystem II complex
When P680 reaction center chlorophyll a dimer absorbs a photon from the LHC of PSII
complex, P680 gets excited to a state P680* at -0.8V. This is equivalent to an energy change 176
KJ/mol and is the difference in energy between the P680 and the excited state, P680* equivalent
to a change in reduction potential of 1.8V. Immediately in 100ps, P680* undergoes a charge
separation, releasing an electron and forming P680+.
16
photon
ps
⎯
⎯→ P680* ⎯⎯→
P680+ + e − .
P680 ⎯⎯
Much of energy of the absorbed light is conserved in P680+. The electron is then transferred to
the bound pheophytin a (Pheo) molecule. The reduced pheophytin a transfers the electron to a
protein bound plastoquinone, QA and then to QB in PSII. Plastoquinone takes up two electrons,
•
one at a time, via a semiquinone intermediate, Q B and two protons from the stroma to form
plastoquinol, QH2 (Figure 10)
− + 2H+
e
Q + e − ⎯⎯→ Q • ⎯⎯
⎯⎯
⎯→ Q H 2
Figure 10: Reduction of plastoquinone to plastoquinol by two successive one-electron
transfers
Plastoquinol is a proton and an electron carrier and is released into the mobile pool of QH2 in the
lipid bilayer of the thylakoid membrane. Plastoquinol diffuses in the membrane and passes its
electrons to cytochrome b6f and releases two protons into the lumen of the thylakoid. The flow of
electron through cytochrome-b6f complex is shown in Figure 11.
17
Figure 11: Electron and proton flow through the cytochrome b6f complex. One electron
from PQH2 passes to the Fe-S center of the Rieske iron – sulfur protein, the other to heme
b of cytochrome b6 and then to the soluble protein plastocyanin
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
From Cyt b6f complex the electrons are taken by Plastocyanin (PC) which is a soluble, mobile,
copper protein in the lumen of the thylakoid. It undergoes reduction to Cu(I) as a result.
2QH2 + 4PC (Cu2+) ⎯⎯→ 2Q + 4PC (Cu+) + 4H+(lumen)
Reduced Plastocyanin passes its electron to P700+ reaction center of Photo system I and is
reoxidised to Cu (II).
The P680+ is one of the most powerful biological oxidants. It extracts electrons from water
molecules. The oxygen evolving complex (OEC), a constituent of PSII oxidizes two molecules
of water to produce 4 electrons, 4 protons and O2. The process is known as photolysis of water,
the net reaction is:
2H2O ⎯⎯→ O2 + 4H+ + 4e—
The OEC has a cluster of Mn atoms, Cl- and Ca2+ at its catalytically active center. Mn complex is
oxidized by P680+ through five oxidation states, So, S1, S2, S3, S4 while removing electrons from
two bound water molecules. So is the most reduced and S4 the most oxidized state. Each
transition from So ⎯⎯→ S4 is a photon (light) driven redox reaction and releases four electrons
18
sequentially from the two H2O molecules bound to OEC. The transition from S4 to SO is light
independent, two more water molecules bind and oxygen is released.
−
−
+
+
−
−
e
e
e
e
S 0 ⎯⎯→
S1 ⎯⎯→
S 2 ⎯⎯→
S 3 ⎯⎯→
S4
H
H
2H+
O2
2H2O
The Mn cluster serves as a charge accumulator and the O2 is formed without the formation of
harmful partly reduced intermediates.
The four electrons released from the water molecules are taken up one at a time by 'Z', a tyrosine
residue161 of D1 polypeptide of PSII and conducted to P680+ to form P680. A single PSII cycles
four times in the productions of one molecule of oxygen from 2 molecules of H2O.
Thus, PSII functions to split water, evolves O2 and release four protons and the cytochrome b6f
complex releases 8 protons into the lumen of the thylakoid.
Electron transfer through photosystem I complex
As in the case of photosystemII, the P700 chlorophyll reaction center of phososystem I absorbs a
photon from its LHC-I and is excited to a state, P700* at -1.3V and charge separation results in
P700+ and an electron. The electron then goes to a chlorophyll acceptor A0 and then to
phylloquinone or vitamin K1 molecule. It finally passes on through various [Fe-S] proteins, Fx,
FA and FB the (Fe-S] cluster from where the electron is taken up by the soluble protein,
ferridoxin, present in the stroma. Thereby, ferridoxin, Fd, is reduced. From reduced ferridoxin,
the electron is transferred to NADP+ by the enzyme ferridoxin-NADP+ oxidoreductase as
follows:
2Fd (red) + H+ + NADP+ ⎯⎯→ 2Fd (oxi) + NADPH
The enzyme ferridoxin NADP+ oxidoreductase takes up a proton (H+) from the stroma to form
NADPH
The oxidized P700+ takes up an electron from PSII via plastocyanin (PC) to regenerate P700
reaction center.
PC (Cu+) + P700+ ⎯⎯→ P700 + PC(Cu2+)
Thus, the main function of PSI is to form NADPH. The two photosystems I and II act in
sequence and are functionally coupled in the linear electron flow from H2O to NADP+ i.e. the
"Z" scheme, where the stoichiometry of the release of O2 is given by the equation:
H2O + NADP+ + ⎯⎯→ ½O2 + NADPH + H+
19
Cyclic electron flow
Depending on the level of NADPH and ATP concentration in the stroma, there is an additional
electron pathway which involves PSI, Plastoquinone (PQ), Cytochrome b6f and Plastocyanin
(PC). When NADPH level is high and NADP+ is low in the stroma, the electrons generated by
illuminated PSI flow to form reduced ferridoxin as usual but since NADP+ is not available, they
return to Cyt b6f complex and thence to PSI via PC (Figure 9. dashed arrow) in a cyclic electron
flow. The absorption of 4 photons by PSI leads to eight protons being pumped by Cyt b6f to
generate proton gradient. Approximately one ATP is generated for 2 absorbed photons or every
two electrons that complete one cycle.
During the cyclic electron flow, no O2 is evolved and no NADPH is formed although some
initial activity of PSII is required to initiate the cycle.
The main function of the cyclic electron flow is to provide ATP for CO2 fixation under
conditions of high NADPH and low ATP. ATP generated as a result of cyclic electron flow is
important in some algae, orchids or under conditions where the PSII activity is low.
Photophosphorylation
The energy of sunlight conserved by phosphorylation of ADP to form ATP during
photosynthesis by chloroplast thylakoid membrance is known as Photophosphorylation just as
the energy released by oxidation of substrates conserved as ATP by mitochondrial inner
membrane is known as oxidative phosphorylation. Thus, both the thylakoid membrane of
chloroplast and the inner membrane of mitochondria are Energy transducers as they transform
one form of energy to another form. In both cases proton gradient or pmf generated by the
electron carriers in the respective membranes drive the synthesis of ATP by ATP SYNTHASE.
ATP Synthase
ADP + Pi ⎯⎯
⎯ ⎯ ⎯⎯→ ATP + H2O
proton gradient
The ATP synthesized during the non cyclic electron flow is known as Non Cylic
Photophosphorylation and that by cyclic electron flow as Cyclic Photophosphorylation.
To recall, during the light reaction of photosynthesis, light induced proton pumping occurs in the
non cyclic electron flow under optimal activities of the PSII and PSI as follows:
1.
2.
During the splitting of water by OEC of PSII, when 4 protons per O2 molecule formed are
released into the lumen of the thylakoid.
As plastoquinone transfers electrons from PSII to Cyt b6f to PC upto 8 protons are pumped
into the lumen of the thylakoid.
As per Mitchell's chemiosmotic theory electron transport and ATP synthesis are coupled by a
proton gradient across the membrane which has 2 components (i) the difference in pH, ∆pH and
(2) difference in charge, ∆ψ.
20
In chloroplasts, the membrane potential across the thylakoid membrane is negligible due to the
transport of Cl- in the same and of Mg2+ in the opposite direction of the membrane to neaturalise
the charge, Thus, the proton gradient between the stroma and the thylakoid lumen generated by
the light reactions drives the synthesis of ATP by ATP synthase.
Andre Jagendorf provided proof for Mitchell's chemiosmotic theory when he showed
experimentally that ATP synthesis can occur in intact chloroplasts in the absence of electron
transport if proton gradient is present. Infact, chloroplasts were incubated at pH4.0 for several
hours. As chloroplast membranes pump protons inwards and not outwards, the inside was at an
acidic pH. These chloroplasts were then quickly transferred to a buffer at pH 8.0 Addition of
ADP and Pi to these chloroplasts generated a burst of ATP synthesis with simultaneous
dissipation of pH gradient. This experiment showed that proton gradient is sufficient to drive the
synthesis of ATP even in the absence of energy input. (Figure 12)
Figure 12: Jagendorf’s experiment showing that chloroplasts synthesize ATP as a result of
the established proton gradient in the absence of energy input.
(Source : Berg, J.M., Tymoczko, J.L. and Stryer, L. 2002. Biochemistry. 5 t h
W.H. freeman and company, New York)
Edition
A brightly illuminated chloroplast generates a pH gradient of 3.5 units across the thylakoid
membrane which corresponds to the stored energy ∆G of about -17 kJ/mol for passage of one
proton. For the reaction,
ADP + Pi ⎯⎯→ ATP, ∆G0’ = 30.5 kJ/mol.
21
The movement of 12 protons representing ~200 kJ of energy can drive synthesis of several
molecules of ATP. In chloroplasts, one ATP is synthesized by ATP synthase for three protons
transported out of the lumen via CF0 into the stroma. Under optimal conditions, noncyclic
electron flow can lead to the synthesis of 3 ATP per O2 evolved, a value which has also been
observed experimentally. However, cyclic electron flow can raise additional ATP for every 2 ethat complete one cycle if required. The ATP formed is released into the stroma.
To summarise, the nonlinear electron flow during LIGHT REACTIONS of photosynthesis
produces 2 NADPH and 3 ATP per O2 molecule released:
2H2O + 2NADP+ + 3ADP + 3Pi ⎯⎯→ 2NADPH + 2H+ + 3ATP + O2
Lateral segregation of the two photosystems
As discussed earlier, PSI and PSII and other protein eomplexes are separate entites and not
contiguous. They have a preferential location in the thylakoid membrane (Figure 4) and are
located 100Å apart. The physical separation or lateral segregation of slow PSII and fast PSI
allows the regulation of the distribution of excitation energy over the two photosystems. This
separation of the two photosystems prevents PSII from becoming a light harvesting antenna of
PSI because the energy level of the excited state of the PSII (P680* relative to ground state
P680) is higher than that of PSI (P700* relative to P700) i.e. ensures that the excitation energy
does not pass directly from PSII to PSI depriving PSII of the excitation energy and maximum
efficiency of photosynthesis is achieved.
The two mobile electron carriers, PQ in the lipid phase of the thylakoid and PC in the thylakoid
lumen can move over long distance between PSII and PSI, enabling the two systems to act in
sequence.
Control of light absorption and electron flow in the two photosystems
The balance in light absorption and electron flow in the two photosystem is important for the
operation of photosynthesis with high efficiency.
The stoichiometry and the antenna size of the LHC associated with the two photosystems alters
under different light conditions. Plants grown in low light have larger antenna size of PSII than
do plants grown in bright light.
The control of electron flow through PSII and PSI is modulated by the level of association of
LHC and the granal stacking of the thylakoid membrane. A hydrophobic region of LHCII
associated with PSII in the thylakoid membrane anchors and binds to a nearby region of the
thylakoid lamellae to form grana (Figure 13). Phosphorylation of a specific threonine in this
hydrophobic anchor region by a kinase results in a change in conformation of LHC-II which
leads to unstacking of the thylakoid lamella. Dephosphorylation by a phosphatase reverses this
effect. The levels of PQH2 and PQ activate the kinase and phosphatase respectively.
When there is normal or bright light (the light has a larger component of blue light) PSII absorbs
more light than PSI and produces PQH2 at a faster rate and PSI is unable to accept electrons at
22
that rate, thus PQH2 accumulates. It activates the kinase which phosphorylates the threonine
residue on LHC-II As a result, unstacking of membranes occur and some of the phosphorylated
LHC-II dissociates from the PSII and moves to the unstacked thylakoid lamella (recall that the
protein complexes can move within the plane of the thylakoid) and binds to PSI to increase the
antenna size of PSI and a larger fraction of light is absorbed by PSI, which can take up more
electrons from PQH2 restoring the balance of electron flow between the two photosystems.
Figure 13: Modulation of granal stacking by light dependent phosphorylation /
dephosphorylation of LHC-II.
(Source: Nelson, D.L. and CoxM (2005) Lehninger Principles of Biochemistry. 4 t h
Edition, W.H. Freeman and company. New York)
Under low illumination (less intense light with a high proportion of long wavelength or red light)
PSI can take up electrons faster from PQH2 than can be supplied by PSII, so that PQ (oxidised)
accumulates. Activation of the phosphatase by PQ leads to dephosphorylation of LHC-Thr P and
the effect of kinase is reversed. LHC returns to PSII and stacking of grana results. Thus,
chloroplasts maintain the balance of electron flow between its two photosystems I and II by a
light dependent feed back mechanism.
Inhibitors of the light reactions
The compounds that interfere with the light Reactions of Photosynthesis are listed in Figure14.
DCMU inhibits close to photosystem II. DMBIB is an antagonist of plastoquinone and inhibits
the cyclie electron flow.
FCCP and DNP are uncouplers of photophosphorylation as they diffuse across the membrane
and dissipate the proton gradient, thus no ATP to synthesized.
Antimycin A interferes with cyclic photophosphorylation. DCCD inhibits the transport of
protons across the thylakoid membrane through CF0-CF1 complex.
Herbicides belonging to atrazine family - Triazines and Diuron specifically bind to D1 protein of
PSII and block the binding of oxidized PQB to the PQ binding site on D1 and all down stream
23
carriers accumulate in their oxidized form, as no electrons are provided to the electron carriers by
PSII. Triazine resistant weeds have a single aminoacid mutation in D1 which renders it unable to
bind the herbicide. This is a major problem in agriculture for destroying these resistant weeds.
Methyl viologen (Paraquat) inhibits the electron flow from PSI to NADP+.
Figure 14: Inhibitors of light reactions of photosynthesis
Bacterial photosynthesis
Photosynthetic bacteria (except cyanobacteria) have only one type of photosystem) whereas
higher plants, algae and cyanobacteria have two photosystem.
The bacterial photosystem is present in cavities called photosynthetic vesicles or
chromatophores of bacterial plasma membrane (Figure 15).
The reaction center of bacteria have been studied in detail and their structure determined by xray crystallography. Much of the present knowledge about the Light Reactions of photosynthesis
has been due to the studies on photosynthetic bacteria.
Bacteria have a simple phototransducing apparatus and their reaction centers are of two general
types. Bacteria, however, contain any one of them.
(a) which is present in the purple bacteria, passes electron from bacteriochlorophyll dimer in the
reaction centre to pheophytin, and then to a quinone (similar to PSII in higher plant, algae
and cyanobacteria)
24
(b) which is present in the green sulfur bacteria passes electron from bacteriochlorphyll to a
quinone and thence to Fe-S center, (similar to PSI in higher plant, algae and cyanobacteria)
Figure 15: Schematic diagram of bacterial photosynthesis
(Source : Rawn, J.D. 1989. Biochemis t ry Neil Patterson Publishers. N. Carolena, USA)
Whereas the electron flow in higher plants, algae and cyacobacteria having two photosystems is
predominantly linear, it is predominantly cyclic in bacteria. However, in green sulfur bacteria,
some electrons also pass in a linear flow to Ferridoxin and then to NAD+ and H2S is the source
of e— to regenerate bacteriochlorophyll instead of H2O in oxygenic photosynthetic organism.
Both the purple bacteria and the green sulfur bacteria use the same set of four modules for
photosynthesis. which are: (1) the reaction center (2)cytochrome bc1 (3) cytochrome C and (4)
bacterial ATP synthase.
Photosynthesis in purple bacteria
The reaction center of purple bacteria such as Rhodobacter capsulata and Rhodobacter
sphaeroides is a large integral membrane protein (100kd) containing 4 polypeptide subunits and
1-3 prosthetie groups, two pairs of the bacteriochlorphylls, pair of bacteriopheophytins, two
quinones, a nonheme iron and 4 hemes in the associated C type cytochrome.
The bacterial
reaction center consists of four polypeptide subunits L, M and H and C where L (31.5 Kd) and M
(34.5 Kd) subunits are more hydrophobic and are paired transmembrance proteins, each
containing five transmembrane α helices whereas H (28.5 Kd) has a single transmembrane αhelix. Most of the H subunit is hydrophilic and lies in the cytoplasmic side of the bacterial
plasma membrane. The fourth protein C2 is a cytochrome which contains four c type hemes and
can diffuse laterally.
The electron carriers involved in the cyclic flow are the bacterial reaction center, a mobile
quinone pool, a cytochrome bc1 oxidoreductase and mobile cytochrome C2. The pathway of
electron flow is very similar to PSII of higher plants, algae and cyanobacteria [Figure 16(a)].
Absorption of light by the special pair of bacteriochloroplyll molecule (P870) is excited (P870*)
and an electron is released forming P870+. This electron goes to a bacteriopheophytion molecule
associated with L subunit and then to adjacent Quinone (QA), which passes the electron to QB
25
converting it to a semiquinone redical. Absorption of a second photon releases another electron
which flows to QB and 2 protons are simultaneously taken up from the cytosol to from quinol
QH2. QH2 is then released from the bacterial reaction center into a mobile pool of quinone. From
QH2 the electron goes to cytochrome bc1 oxidoreductase and then to cytochrome C2. The
cytochrome bc1 oxidoreductare (100 Kd) is a membrane spanning protein containing cytochrome
b, cytochrome c1 and a Rieske iron sulfur protein. Its function is analogous to cytochrome b6f
complex of plants, algae and cyanobacteria and complex III of mitochondria. Cytochrome bc1
couples the flow of electrons from quinol to cytochrome C2 with transport of proton into the
intravesicular space to generate proton gradient.
1.0
P840*
P870*
Fe
e—
e—
NADP+
Q
– 0.5
E’0 (volts)
NAD—
Pd-NAD
reductase
Pheo
Excitons
Q
0
0.5
Excitons
RC
P870
Cyt
bC1
complex
Cyt
C2
Purple bacteria
(pheophytin-quinone type)
(a)
RC
P840
Cyt
C553
Cyt
bC1
complex
Proton
gradient
Proton
gradient
Green sulfur bacteria
(FE-S type)
(b)
Figure 16: The flow of electron through the photosynthetic modules of – (a) purple
photosynthetic bacteria and (b) green sulfur bacteria
(Source: Ne lson, D.L. and CoxM (2005) Lehninger Principles of Biochemis t ry. 4 t h
Edition, W.H. Freeman and company. New York)
The reduced cytochrome C2 diffuses laterally to bacterial reaction center where it transfers its
electron to the positively charged special pair of reaction center (P870+) to form P870. Thus,
light energy is converted and stored into chemical energy in the form of proton gradient which
drives the synthesis of ATP by bacterial ATP synthase.
Photosynthesis in green sulfur bacteria
It involves Reaction center, Cytochrome bc1 and cytochrome C6 and bacterial ATP synthase but
the process is different and involves additional enzymatic reaction [Figure 16(b)]. The excitation
of reaction center P840 to P840* releases an electron and forms P840+ radical. The electron
flows through quinone to cytochrome bc1 and then via cytochrome C in a cyclic flow to P840* to
regenerate P840 with associated proton pumping which generates proton gradient. Also, some
electrons from P840*, go to a [Fe-S] protein, ferridoxin and then via ferridoxin-NAD reductase
26
to NAD+ to form NADH. Oxidation of H2S releases H2, S and electrons which replace electrons
taken from the reaction centre P840* to reduce NAD+. This step is analogous to the oxidation of
H2O by oxygenic plants. The overall reaction, in CO2 fixation to synthesize sugars is given by:
+
light
H2S + H+ + NAD+ ⎯⎯
⎯→ 2H + S + NADH
light
12H2S + 6CO2 ⎯⎯
⎯→ C6H12O6 + 12S + 6H2O
Some phososynthetic bacteria use H2 or other organic compound as an electron source for the
light dependent reduction of CO2 (Table 1).
Why are there two photosystems in higher plants, algae and cyanobacteria?
Purple and green bacteria have only one photosystem. They use H2S or H2 i.e. compounds with
lower (more negative) reduction potential as a source of electrons and reduce NAD+ to NADH.
The reaction is
H2S ⎯⎯→ S + 2H+ + 2e- [E0 = -0.25V]
The two Photosystem organisms use water as an electron donor where the reactions are:
H2O ⎯⎯→ 2H+ + 2e- + 1/2 O2
[E0 = + 0.815V], and
NAD+ (P)+ + H+ + 2e- Æ NAD (P) H-; [E0 = -0.32]
Thus, less energy is required to boost electrons from H2S to a level sufficient to reach the
reduction potential of NAD+ than that to boost electrons from H2O to NAD(P)+. The former is
possible by a single photon absorbed by one PS in bacteria.
In higher plants and algae the absorption of a single photon by P680 cannot boost e- to the level
required to reduce NADP+ directly and the energy is also used to stabilize the earlier reactive
intermediates. Hence, both PSII and PSI are coupled functionally and each acts linearly to boost
electrons part of the way to reduce NADP+.
Dark reactions
As mentioned earlier, the synthesis of carbohydrates [CO2 fixation] occurs in the stroma in a
series of reactions, called the dark reactions as they do not require sunlight, although some of the
reactions are accelerated in the presence of light.
Melvin Calvin, Andrew Benson and James A. Bassham had worked out the sequence of the dark
reactions in growing cultures of algae between 1946 to 1953. Calvin showed that the first
product of CO2 fixation is 3-Phophogycerate by using 14CO2. These reactions are named after
him. He received Noble prize in 1961 for his work in this field.
Calvin and his group showed that two molecules of 3-Phosphoglycerate are formed from
ribulose 1,5bisphosphate and one molecule of CO2 is incorporated during this reaction, catalyzed
by the enzyme Ribulose 1,5bisphosphate carboxylase / oxygenase, Rubisco, in stroma. Further
studies by Calvin’s group showed that 3-phosphoglycerate is converted back to ribulose
27
1,5bisphosphate by several enzymes. This set of reactions is called Reductive Pentose cycle or
Calvin cycle or photosynthetic carbon reductive cycle. Since the first intermediate in the Calvin
cycle is a C3 compound, it is also called the C3 cycle.
In animal cells, the set of reactions used in the conversion of pentose phosphates to hexose
phosphates during the nonoxidative phase of pentose phophate pathway are mostly used in the
Calvin Cycle but in the opposite direction to convert hexose phosphate to pentose phosphate.
Calvin Cycle in addition uses several enzymes which make the Reductive Pentose cycle
irreversible. All the enzymes involved in this Photosynthetic Carbon Reduction or Calvin Cycle
are present in the stroma.
Ribulose 1, 5 bisphosphate carboxylase / oxygenase (Rubisco), sedoheptulose 1, 7 bis
phosphatase and Ribulose -5 phosphate Kinase are the three enzymes unique to photosynthetic
organisms and are absent in animals.
Calvin cycle
The reactions of the Calvin cycle and the enzymes involved are given in Figure 17 and Figure
1 8. Calvin cycle is discussed in 3 phases:
Phase I: CO2 Fixation
Carbon dioxide (CO2) is added to an acceptor, Ribulose 1,5 bis phosphate (RuBP) to give 2
molecules of 3-Phosphoglycerate (3-PG) by the enzyme Ribulose 1,5 bisphosphate
carboxylase/oxygenses or Rubisco. This is the most abundant protein in plants, and probably on
earth, about 15% of chloroplast protein (1 of Figure 17).
Phase II (a) : Reduction of 3 Phosphoglycerate
The 3-Phosophogylcerate is phosphorylated by ATP to form 1,3 bisphosphoglycerate. The
1,3 bisphosphogylcreate is reduced by NADPH to form glyceraldehydes 3- phosphate (2 and 3 of
Figure 17). The Glyceraldehyde 3-phosphate is an important intermediate which can (a) be
converted by a series of enzymes to form (i) Hexose, starch, sucrose and (ii) Ribulose 1,5 bis
phosphate or (b) produce energy via glycolysis.
Fixation of 1CO2 results in the formation of 2 molecules of glyceraldehydes-3-phosphate and 2
molecules of ATP are hydrolysed and 2 molecules of NADPH oxidized.
Thus, fixation of 6 molecules of CO2 forms 12 molecules of Glyceraldehyde -3-Phosphate (G-3P) and requires 12 ATP and 12 NADPH.
At this stage, the pathway splits to make 1 molecule of Hexose and to regenerate the acceptor
molecule, RuBP.
Phase II (b) : Formation of Hexose
Of the 12 molecules of G-3-P, two G-3-P are converted to fructose-6-phosphate (4 to 6 of Figure
17) as follows:
28
(i)
One glyceraldahyde-3-phosphate is converted to dihydroxyacctone phosphate (DHAP)
by Triose phosphate isomerase.
(ii) One molecule of glyceraldhyde-3-phosphate and one of dihydroxyacetone
phosphate are condensed to form fructose1-6 diphosphate by the enzy me
aldolase.
(iii) Fructose 1-6 diphosphate is hydrolysed by fructose diphosphatase in an irreversible
reaction to give fructose-6-phosphate which is converted by a series of
enzy me reactions to form (a) Glucose-1-phosphate and (b) Ribulose 1-5 bis
phosphate (Phase III).
(iv) Fructose-6-phosphate by the action of the enzy me Phosphoglucoisomerase
forms Glucose-6- phosphate.
(v) Glucose-6-phosphate is converted by phosphoglucomutase to Glucose-1phosphate which is the precursor of (a) disaccharide, sucrose, synthesized in
the cytosol and (b) poly saccharide, starch synthesized in the stroma.
These reactions are given in (Figure 18). Thus, 2 molecules of G-3-P are
converted to fructose-6-phosphate or Glucose-1- phosphate.
Phase I Carboxylation.
1 6-Ribulose 1,5-diphosphate + 6CO2 + 6H2O 12 3-phosphoglycerate (enzyme; ribulosc diphosphate
carboxylase).
Phase II Reduction of phosphoglycerate
2 12 3-Phosphoglycerate + 12ATP 12 1,3-diphosphoglycerate + 12ADP (phosphoglycerate kinase)
3 12 1,3-diphosphoglycerate + 12NADPH + 12H+ 12 glyceraldehyde 3-phosphate (NADPglyceraldehyde 3-phosphate dehydrogenase).
Phase III Regeneration of CO2 acceptor
4 5 Glyceraldehyde 3-phosphate ' 5 dihydroxyacetone phosphate (triose phosphate isomerase)
5 3 Glyceraldehyde 3-phosphate + 3 dihydroxyacetone phosphate ' 3 fructose 1, 6-diphosphate
(aldolase)
6 3 Fructose 1,6-diphosphate + 3H2O 3 fructose 6-phosphate + 3Pi (fructose diphosphatase)
7 2 Fructose 6-phosphate + 2 glyceraldehyde 3-phosphate ' 2 xylulose 5-phosphate + 2erythrose 4phosphate (transketolase).
8 2 Erythrose 4-phosphate + 2 dihydroxyacetone phosphate ' 2 sedoheptulose 1, 7-diphosphate
(aldolase)
9 2 Sedoheptulose 1, 7-diphosphate + 2H2O 2 sedoheptulose 7-phosphate + 2Pi (sedoheptulose
diphosphatase)
10 2 Sedoheptulose 7-phosphate + 2 glyceraldehyde 3-phosphate ' 2 ribose 5-phosphate + 2 xylulose 5phosphate (transketolase)
11 2 Ribose 5 – phosphate ' 5-ribulose 5-phosphate (ribose phosphate isomerase)
12 4 xylulose 5-phosphate ' 4 ribulose 5-phosphate (ribulose phosphate epimerase)
13 6 ribulose 5-phosphate + 6ATP 6 ribulose 1, 5 diphosphate + 6ADP (phosphoribulokinase)
Figure 17: Reactions of the Calvin cycle and the enzymes involved are shown in parenthesis
29
Figure 18: Calvin cycle depicting the CO2 fixation, Reduction and Regeneration phases
which occur in the stroma and its relationship with the sucrose synthesis in the cytosol
(Source : Lodish, H., Baltimore, D., Berk, A., Zipursky, S.L., Metasudaira, P. and
D arnell, J. 1995. Molecular cell Biology, 3 r d Edition, Scientific American Books W.H.
freeman and company, USA)
30
Phase III : Regeneration of RuBP
The remaining 10 molecules of G-3-P are used to regenerate 6 molecules of RuBP by a
complicated set of reactions (4 t0 13 of Figure 17). Of the 10 molcule of Glyceradehyde -3Phosphate,
1.
2.
3.
4.
5.
6.
7.
8.
9.
Four molecules are converted to Dihydroxyacetonephosphate.
There after, two molecules each of glyceraldehyde-3-phophate and Dihydroxy acctone
phophate form 2 molecules of Fructose--6 phosphate by aldolase.
Then, 2 molecules of glyceraldehyde-3-phosphate and 2 molecules of Fructose -6phosphate form 2 xylulose -5- phosphate and 2 of erythrose-4- phosphate by the enzyme
transketolase which requires the coenzyme thiamin pyrophosphate.
Two molecules erythrose-4-phosphate and two of dihydroxyacetonephosphate are
converted to 2 molecule of seduheptulose 1,7 bis phosphate by aldolase.
Sedoheptulose 1,7 bisphosphate is hydrolyzed by seduheptulose bis phosphatase to form
sedoheptulose -7- phosphate and Pi.
Two molecules of Sedoheptulose-7-phosphate and 2molecules of glyceraldehydes-3phosphate form 2 molecules of xylulose-5-phosphate and 2 molecules of ribose-5phosphate by Transketolase.
Four molecules of xylulose-5-phosphate are converted to four of Ribulose-5-phosphate
by ribulose epimerase
Two molecules of ribose-5-phosphate are converted to 2 molecules of ribulose-5phosphate by isomerase reaction.
Six molecules of ribulose-5Phosphate is then phosphorylated by ATP dependent
phosphoribulo kinase to form Ribulose-1-5 bis phosphate.
Thus, for the synthesis of 1 Hexose from 6CO2, 12 molecule of NADPH and 18 molecules of
ATP are required in the Calvin Cycle and the net reaction of the Calvin cycle is.
6 CO2 + 18 ATP + 12 NADPH + 12H2O ⎯⎯→ C6H12O6 + 18 ADP + 18Pi + 12NADP+ + 6H+
Bio Synthesis of Starch and Sucrose
Starch and sucrose are the major storage sugars in plants.
(a) Synthesis of starch
Starch is a polymer of glucose residues but is less branched than the animal storage sugar,
glycogen. It is stored in chloroplasts and synthesized from the activated precursor ADP-glucose
in the stroma.
Fructose 6-phosphate is converted to Glucose-1-phosphate (Stage IIb of Calvin cycle) which
reacts with ATP to form ADP-glucose and pyrophosphate (PPi) by the enzyme ADP: glucose
Phosphorylase. Hydrolysis of PPi to 2Pi by pyrophosphatase renders the entire reaction
irreversible. The glucose unit of ADP-glucose is then transferred to the polysaccharide chain, to
form starch.
31
ATP + glucose-1-phosphate ⎯⎯→ ADP-glucose + PPi
PPi ⎯⎯→ 2Pi
ADP-glucose + (glucose)n ⎯⎯→ (glucose)n+1 + ADP
(b) Synthesis of Sucrose
Sucrose, a disaccharide, is synthesized in the cytosol. Hexose phosphates cannot be transported
across the chloroplast membranes. However, a triosephosphate-Pi antiporter transports triose
phosphate across the inner membrane of the chloroplast to the cytosol for an obligatory exchange
of Pi into the stroma (Figure 18).
The triose-phosphates are converted by the cytosolic isozymes of the stromal enzymes by similar
reactions to form fructose-6-phosphate and glucose 1-phosphate which is converted to its
activated precursor UDP-glucose. Fructose-6-phosphate then reacts with UDP-glucose to form
sucrose-6-phosphate by the enzymes sucrose synthetase. Sucrose-6-phosphate is hydrolysed
to sucrose (Figure 18) which is exported to other parts of the plant through the veins.
Rubisco
This is the first enzyme in the CO2 fixation and is unique to the Dark reactions and the most
important enzyme since nearly all life on the earth ultimately depends on its action. It comprises
almost 50% of the leaf protein and is the most abundant protein in the biosphere. This enzyme is
estimated to be synthesized at the rate of about 4x109 ton/y which fixes about 1011 tons of CO2/y.
Rubisco (560 Kd) from higher plants and photosynthetic bacteria consists of 8 large (L) subunits
(56 Kd) encoded by chloroplast DNA and 8 small (S) subunits (14 kd) specified by a nuclear
gene. The enzyme from certain photosynthetic bacteria is a L2 dimer where the L subunit is
structurally similar to that of the L8S8 enzyme. The function of small subunit is not known
Each L subunit contains the catalytic and the regulatory site. The enzyme has 8 catalytic sites
and is active in the absence of S subunit The 8 S subunits are arranged in two tetramers near the
top and the bottom of the molecule. The 8 large subunits are arranged between the two tetramers
of small subunits.
X-ray studies by Carl-Ivor Branden and by David Eisenberg have shown that L8S8 enzyme has
the symmetry of a square prism.
The enzyme Rubisco has a low catalytic efficiency, k cat of 3 s-1. The carboxylation reaction
catalyzed by Rubisco is shown in (Figure 19).
The slow step of the reaction is the enolization to form Enediol Intermediate. The CO2 reacts
with the Enediol Intermediate to from 2 carboxy-3-Ketoarabinitol 1,5 bisphosphate (CKAB).
Water then reacts with 3-keto carbon, to form an adduct which cleaves to give a molecule of 3phosphoglycerate (3 PG) and a carbanion intermediate. The stereospecific protonation of the
carbanion intermediate gives the second molecule of 3PG.
32
Figure 19: Carboxylase activity of Rubisco
Rubisco is inhibited by its β-keto acid intermediate analogs, 2-carboxyara binitol-1-phosphate
(CAIP) and 2 carboxy-arabinitol-1,5, bisphosphate (CABP), the competitive inhibitors.
33
CH 2 OPO 32−
CH 2 OPO 32−
HO − C − CO 2−
HO − C − CO 2−
H − C − OH
H − C − OH
H − C − OH
H − C − OH
CH 2 OH
2-Carboxyarabinitol1-phosphate (CA1P)
CH 2 OPO 32−
2-Carboxyarabinitol1-5-biophosphate (CABP)
This enzyme can also catalyse an oxygenase reaction which will be described in a later section.
(2.2.3 Photorespiration)
Regulation of Calvin cycle
Calvin cycle reactions of photosynthesis are dependent on ATP and NADPH which are
produced by the Light Reactions. Regulation is achieved at two levels by modulation of (i)
Rubiso, the first enzyme catalyzing the rate limiting step and (ii) other critical enzymes of the
Calvin cycle.
Regulation of Rubisco
Rubisco is activated by pH, CO2 and Mg2+. During photosynthesis, the rate of CO2 uptake is
determined by availability of CO2 at the site of Carboxylation, the supply of Ribulose 1,5
bisphosphate and activation state of Rubisco.
Pumping of protons during the light reactions leads to an increase in the pH of stroma to 7.5 to
8.0 and Mg2+ also enters to balance the positive charge due to loss of H+. Rubisco is carboxylated
by CO2 which is not involved in catalysis. This CO2 binds to the amino group of a lysine residue
on the enzyme surface. A molecule of Mg2+ in turn binds to CO2, thus activating the enzyme.
(Figure 20) This activation by CO2 and Mg2P results in the increase in maximum rate (Vmax) of
the enzyme reaction and in the affinity of the enzyme for CO2 and is mediated by a soluble
enzyme, Rubisco activase.
The competitive inhibitor, 2 carboxy arabinitol-1-phosphate (CAIP) is synthesized at night
by some plants. The CAIP binds to the catalytic site and prevents carboxylation of ribulose 1,5
bis phosphate at night. Rubisco activase facilitates the release of CAIP from Rubisco.
Regulation of other enzymes of Calvin cycle
(a) Light dependent activation of Calvin cycle enzymes
Glyceraldehyde-3-phosphate dehydrogenase, Fructose 1,6bisphosphatase Seduheptulose1,7bis phosphatase, and Phosphoribulokinase are activated by reduced thioredoxin by sulfhydryl
exchange reaction. Thioredoxin is a small protein which has two SH groups. These -SH groups
can be easily oxidized to form disulfide [S-S-] bond. Thioredoxin is in turn activated by reduced
ferridoxin by the enzyme ferridoxin-thioredoxin reductase. When the chloroplasts are strongly
illuminated, the NADP+ is converted to NADPH. Thus, NADP+ is not available for ferridoxin-
34
NADP+ Reductase leading to the accumulation of reduced ferridoxin. The high levels of reduced
ferridoxin activates thioredoxin which in turn activates the Calvin cycle enzymes mentioned
above (Figure 21).
In addition, some of the enzymes, Phosphofructokinase (enzyme of glycolysis) and Glucose-6phosphate dehydrogenase (enzyme of Hexose mono phosphate shunt) which can interfere in the
Calvin cycle reactions by withdrawing or acting on fruclose-6-phosphate and Glucose-6Phosphate respectively are inhibited by reduced thioredoxin formed in presence of sunlight.
Figure 20: Activation of Rubisco
(b) Also, activities of fructose 1,6 bisphosphatase and sedoheptulose 1,7 bis phosphatase
are dependent on pH and Mg2+. Regulation also occurs by change in the ionization state of
effector molecule, phosphoglycerate (PG) for phosphoribulokinase, PG2- inhibits the
phosphoribulose kinase, but PG3- does not. Alkalinization of stroma (pumping out of proton)
favours PG3- formation leading to the reversal of inhibition by PG2-.
There is also evidence that the genes of chloroplasts are regulated at the transcriptional level and
are repressed by sucrose and glucose.
35
Figure 21: Light dependent regulation of Calvin cycle enzymes: Glyceraldehyde-3phosphate dehydrogenase, fructose 1, 6-bisphosphatase, sedoheptulose 1, 7-bisphosphatase
and phosphoribulokinase
Efficiency of photosynthesis
During the Linear electron flow or Z scheme of photosynthesis, PSII cycles four times i.e.
absorbs 4 photons to form one molecule of O2 and release 4 electrons which go to PSI. PSI
absorbs another 4 photons to reduce NADP+. A total of 8 photons are thus required to synthesize
2 molecules of NADP and 3 of ATP per O2 evolved. This agrees with the observed experimental
value of 0.12 O2 release per absorbed photon, when both PSI and PSII are operating at optimal
efficiency. Thus, 48 photons must be absorbed for 12 NADPH and 18 ATP required for the
synthesis of a molecule of hexose in the dark reactions. If the cyclic photophosphorylation also
operates then the number of photons needed will be larger.
Forming a mole of hexose from CO2 and H2O requires 2870 kJ of energy. As the energy input of
photons is dependent upon the wavelength of light used, assuming that light of 650nm
wavelength is absorbed, then 48 photons of such light will correspond to about 8200 kJ of
energy. Therefore, the efficiency of photosynthesis is around 35%. This is in agreement with
direct experimental results under optimal conditions for photosynthesis.
36
Photo respiration
Rubisco is a bifunctional enzyme and has an associated oxygenase activity It catalyzes the
oxygenation of Ribulose 1,5 bisphosphate (RuBP) at the same catalytic site on the L subunit as
the carboxylation where carbondioxide and oxygen are the classical competitive inhibitors. Both
the enzyme activities are inhibited by 2-carboxyarabinitol 1,5, bisphosphate, CABP. The Km for
CO2 is 12µM as against 250µM for O2 i.e. Km for O2 is much higher than that for CO2.
When O2 concentration is high, Rubisco catalyzes the reaction of O2 with Ribulose 1,5 bis
phosphate to form phosphogycolate and 3-phosphoglycerate in the chloroplast (Figure 22)
Phosphoglycolate is toxic to plants being a powerful inhibitor of Triosephosphate Isomerase (an
important enzyme of glycolysis and Calvin cycle). It is dephosphorylated to glycolate by a
phosphoglycolate phosphatase. The oxygenase activity and the glycolate salvage pathway
consuming O2 and releasing CO2 is called Photorespiration or C2 cycle and occurs in three
cellular compartments-chloroplasts, mitochondria and peroxisomes.
Figure 22: Oxygenase activity of Rubisco
Glycolate passes on to peroxisomes where it is oxidized to give hydrogen peroxide and
glyoxylate. Hydrogen peroxide being toxic is acted upon by catalase. Glyoxylate is converted to
glycine which then enters the mitochondria where two molecules of glycine form one molecule
each of serine, CO2 and NH3. The latter two products are released and serine is returned to the
peroxisome where it is converted to glycerate. Glycerate then enters the chloroplast, where it is
converted by ATP dependent glycerokinase to 3-phosphoglycerate (3PG) which enters the
Calvin cycle (Figure 23).
Thus, photorespiration is a light dependent reversal of the CO2 fixation which oxidizes RuBP,
uses ATP, and is a wasteful process. About 25% of the (RuBP) is lost and 75% is available as
3PG. Infact, it decreases the efficiency of photosynthesis. Photorespiration occurs at all times to
some extent in C3 plants but is very active during such conditions as bright sunlight, high
temperatures, low CO2 and high O2 concentrations. Early during evolution when the
photosynthesis evolved, CO2 content was high and O2 was neglible in the atmosphere and the
effect of O2 on Rubisco activity was not perceptible. However, as the O2 content in the
37
Figure 23: The photorespiration pathway for salvage of glycolate produced by oxidation of
ribulose 1,5 bisphosphate (RuBP) by Rubisco
(Source : Rawn, J.D. 1989. Biochemistry Neil Patterson Publishers. N. Carolena, USA)
38
atmosphere increased, photorespiration assumed significance. Inspite of its deleterious effects,
why has the oxygenase activity of Rubisco been retained over the years during evolution? No
Rubisco has been found that has no oxygenase activity. One explanation suggests that when CO2
concentration is low, but the sunlight is sufficient, the light reaction proceeds as usual to form
ATP and NADPH which accumulate, as they are not utilized in the Calvin cycle reactions due to
low availability of CO2. This, in turn, leads to an increase in the reduced electron carriers, proton
gradient across the thylakoid membrane and photochemical excited states of the phostosystems,
which have deleterious effects on the Photosynthetic assemblies. Photorespiration by consuming
ATP and NADPH, provides ADP and NADP+ for the activity of the light reaction to continue i.e.
help in dissipating the photochemical energy and thus prevents damage to the photosynthetic
components.
This hypothesis is supported by the observation that bright illumination of leaves or chloroplasts
in the absence of both CO2 and O2 results in the rapid and irreversible loss of their
photosynthetic ability.
Some plants have bypassed the harmful effects of photorespiration by using the C4 and CAM
PATHWAY which conserve CO2 as C4 intermediates (oxaloacetate, malate etc.) This is different
from the Calvin cycle or the C3 pathway which uses C3 intermediates.
The C4 and CAM pathways are two evolutionary solutions for maintaining photosynthesis on
hot, dry days.
C4 Pathway
C4 pathway is also known as Hatch-Slack pathway after the scientists who elucidated this
pathway in 1960s. The C4 cycle operates in several thousand species such as sugarcane, corn,
millet - the agriculturally important plants and in tropical plants exposed to intense sunlight and
high temperatures and dryness.
In C4 plants, there are two distinct types of photosynthetic cells (1) Bundle Sheath cells and (2)
Mesophyll cells
As discussed in earlier section, CO2 fixation in the C3 plants occurs in the mesophyll cells.
However in C4 plants, the Calvin cycle photosynthesis occurs in specialized bundle sheath cells
lying below the mesophyll cells in the interior of the leaf and are protected from atmospheric
oxygen (Figure 24).
The bundle sheath cells contain chloroplasts and Rubisco but no phosphoenol pyruvate (PEP)
carboxylase enzyme. It has less grana and more starch in their chloroplasts than those of C3
plants and are unusual as they lack PSII and contain only PSI which carries out cyclic electron
flow, and therefore no O2 is evolved.
The meseophyll cells are exposed to the outside, do not contain Rubisco but have
phosphoenolpyruvate (PEP) carboxylase which captures CO2. The C4 pathway physically
separates the CO2 capture from the Calvin cycle reactions in C4 plants. The C4 pathway is
shown in Figure 25.
39
Figure 24: Diagram of the structure of leaves of C4 and C3 plants
(Source : Rawn, J.D. 1989. Biochemistry Neil Patterson Publishers. N. Carolena, USA)
Figure 25: The C4 pathway involving the capture of CO2 in the mesophyll cells and its
transfer to the bundle sheath cells for fixation by Calvin cycle
(Source : Lodish, H., Baltimore, D., Berk, A., Zipursky, S.L., Metasudaira, P. and
Darnell, J. 1995. M olecular cell Biology, 3 r d Edition, Scientific American Books W.H.
f reeman and company, USA)
40
Carbon dioxide is fixed in the mesophyll cells by PEP carboxylase catalyzed reaction between
HCO-3 (which is in equilibrium with CO2) and phosphoenol pyruvate (PEP) to form
oxaloacetate. This enzyme has a very low Km for CO2 and has no oxygenase activity, so that
even at low CO2 concentration oxaloacetate is formed. Oxaloacetate can be converted to any of
the other C4 acids. Malic acid is formed by reduction of oxaloacetate with NADPH specific
malic dchydrogenase, and transported to the bundle sheath cells through plasmodesmata where
malic enzyme catalyzes its oxidative decarboxylation to from pyruvate and CO2. The CO2
released enters the Calvin eycle and the pyruvate is transported to the mesophyll cells where it is
converted to PEP by pyruvatephosphate dikinase using ATP to form AMP and pyrophosphate.
The pyrophosphate is further hydrolysed to give two Pi. The AMP conversion to ADP requires
another molecule of ATP. So that two molecules of ATP are required in the conversion of
pyruvate to PEP. The species of C4 plants which have PEPcarboxy kinase use only one ATP. The
CO2 fixation, therefore, consumes 4-5 ATP in C4 plants instead of 3ATP in Calvin cycle per CO2
molecule fixed. Thus, the C4 pathway is energetically more expensive and the overall efficiency
of photosynthetic production of sugars is lower than that in C3 plants. However, the net rate of
photosynthesis in C4 plants can be two to three times of that in C3 plants. It is observed that the
C4 plants flourish in hot, dry, tropical climate whereas C3 plants predominate in temperate
climate (low temperature).
Crassulacean acid metabolism (CAM)
The CAM pathway is evolved in sacculent (water storing) plants e.g. cacti, pineapple and
members of the Crassulacean family. In these species, the stomata close during the day and open
during the night, as the temperature is lower at night and also humidity is usually higher. This
helps the dessert plant to conserve water and reduce transpiration (prevent water loss during the
day). These plants take up CO2 at night, convert it into malic acid or isocitric acid (C4
compounds). During the day, when stomata are closed, CO2 is released from these C4 organic
acids and is used immediately in the Calvin cycle reaction. This process is known as
Crassulacean acid metabolism (CAM) and the plants that employ it are known as CAM plants. It
is analogous to the C4 pathway but in this case there is no physical separation of CO2 capture
and Calvin cycle. The two processes occur at different times (night/day).
The C4 and CAM plants eventually use Calvin Cycle, like the C3 plants to make sugar from
CO2.
Suggested Readings
1.
Barber. J., and Anderson, B., 1994. Revealing the blue print of photosynthesis. Nature 370, 31-34.
2.
Bassham, J. A., 1962. The path of carbon in photosynthesis Sci. Am. 206, 88 – 100.
3.
Buchanan, B.B., 1991. Regulation of CO2 assimilation in oxygenic photosynthesis: the ferridoxin/thioredoxin
system. Perspective on its discovery, present status and future development. Arch. Biochem. Biophy., 288, 19.
4.
Green, B.R., Pichersky, E, and Kloppstech, K, 1991. Chlorophyll a/b binding proteins: an extended family.
Trends Biochem. Sci. 16, 181-186.
5.
Govindjee and W.J. Coleman, 1990. How plants make oxygen. Sci. Am., 262, 50-58.
41
6.
Halliwell, B. 1981. The structure and function of chloroplast in green leaf cells. Clarendon Press. Oxford.
7.
Hatch, M.D. 1977. C4 pathway photosynthesis: Mechanism and physiological function. Trends Biochem. Sci.,
2, 199-201.
8.
Hatch, M.D. and Boardman, N.K., (Eds) 1981. The Biochemistry of Plants, Vol. 8. Photosynthesis Academic
Press. New York.
9.
ibid (1987). Vol 10. Phtosynthesis.
10.
Jagendorf. A.T., 1967. Acid-base transitions and phosphorylation by chloroplasts. Fed. Proc., 26, 1361-1369.
11.
Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W., and Krauss, N., 2001. Three Dimensional
structure of cyanobacterial photosystem I at 2.5Å. Nature 411, 909-917.
12.
Junge, W., Lill, H., and Engelbrecht, S., 1997. ATP synthase. An electrochemical transducer with rotatory
mechanics. Trends Biochem. Sci. 22, 420-423.
13.
Kargul, J., Nield, J. and Barber, J., 2001. Three Dimensional reconstruction of a light harvesting complex IPhoto system I (LHC I – PSI) super complex from the green alga Chlamydomonas reinhardtii J. Biol. Chem.
278, 16,135 – 16,141.
14.
Knaff. D.B., 1989. Structure and Regulation of ribulose–1,5–bisphosphate carboxylase/ oxygenase. Trends
Biochem. Sci. 14, 159-160.
15.
ibid, 1990. the cytochrome bc1 complex of photosynthetic bacteria. Trends Biochem. Sci. 15, 289-291.
16.
ibid 1991. Regulatory phosphorylation of chloroplast antenna proteins. Trends Biochem. Sci. 16, 82-83.
17.
McCarty, R.E., and Hammes, G.G. (1987). Molecular Architecture of chloroplast coupling factor. Trends
Biochem. Sci. 12, 234-237.
18.
Miziorko, H.M. and Lorimer, G.H., 1983. Ribulose 1, 5-bisphosphate carboxylase-oxygenase. Annu. Rev.
Biochem. 52, 507-535.
19.
Morris, E.P., Hankamer, B, Zhelva, D., Friso, G., Barber, J., 1997. The three dimensional structure of a
photosystem II core complex determined by electron crystallography. Structure 5, 837-849.
20.
O’Keefe, D.P., 1988. Structure and function of the chloroplast bf complex. Photosynthetic Research, 17, 189216.
21.
Richter, M.L., Hein, R., and Huchzermeyer, B. 2000. Important subunit interactions in the chloroplast ATP
synthase. Biochim. Biophys. Acta., 1458, 326-329.
22.
Rutherford, A.W., 1989. Phtosystem II, the water splitting enzyme. Trends Biochem. Sci., 14, 227-242.
23.
Scheller, H.V. and Maller, B.L. 1990. Photosystem I polypeptides. Physiologia Plantarum, 78, 484-494.
24.
Ting, I.P., 1985. Crassulacean acid metabolism. Annu. Rev. Plant Physiol., 36, 595-622.
25.
Zuber, H., 1986. Structure of light harvesting antenna complexes of photosynthetic bacteria, cyanobacteria and
red algae. Trends Biochem. Sci. 11, 414-419.