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INTERMEDIARY METABOLISM

Photosynthesis

Dr. (Mrs.) Veena Taneja Professor of Biochemistry (Superannuated)

Banaras Hindu University Varanasi - 221 005

02 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 Photosynthesis , Chloroplas t , Light React ions , Photosystem I , Photosystem II , Light Harvest ing Complex, ATPSynthase, Photophosphorylat ion, Dark React ions, Calvin Cycle, Rubisco, C3- and C4- Pathways, CAM

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Photosynthesis is the process where by many plants, algae and microorganisms synthesize

carbohydrates from carbon dioxide and water using the energy of the sun:

6CO2 + 6H2O C⎯⎯ →⎯ light6H12O6 + 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).

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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 edit ion. 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

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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.

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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 Edit ion, 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

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power NADPH in a series of reactions occurring in the thylakoid membranes known as LIGHT REACTIONS. 12H2O + 12 NADP+ + 18ADP + 18P2 12 NADPH ⎯⎯ →⎯light + 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 [CH⎯→⎯ 2O] + 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 Reductant React ion Plants , a lgae, cyanobacter ia H2O CO2 + 2H2O [CH⎯→⎯ 2O] + H2O + O2

Green sulfur bacter ia H 2S CO2 + 2H2S [CH⎯→⎯ 2O] + H2O + 2S Purple sulfur bacter ia ]HSO[ 3

− CO2 + H2O + 2 [CH]HSO[ 3− ⎯→⎯ 2O] + 2 ]HSO[ 4

−

Nonsulfur photosynthetic bacter ia

H2 or many other reductants such as lacta te

CO2 + 2H2 ⎯→⎯ [CH2O] + H2O

CO2 + 2 HC — OH [CH⎯→⎯ 2O] + H2O + 2 C = O

CH3

−COO

CH3

−COO

Pyruvate Lactate

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)

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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 H2

18O and unlabelled CO2 that neither of the oxygen atoms come from CO2 and that one of the oxygen from CO2 ends up in carbohydrate. CO2 + 2H2

18O [CH⎯⎯ →⎯light2O] + H2O + 18O2

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. 4Fe3+ + 2H2O 4Fe⎯⎯⎯⎯⎯ energylight → 2+ + 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).

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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 Edit ion, 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) 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.

(2) A small protein, Psa C (9 kd) which contains Fe-S centers. (3) Extrinsic protein Psa F binds soluble plastocyanin, a mobile electron carrier, to the

lumenal side of PSI. (4) Psa D, an extrinsic protein, binds ferridoxin to the stromal side of PSI. (5) Small peripheral proteins, PsaE, PsaI to M and few others.

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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 α-helices 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) 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.

(2) 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)].

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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. 5t h Edit ion 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 edit ion. 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)

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(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. 5t h Edit ion 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 400-650nm) 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) Cytochrome f (cyt f), (where f is frons, French for leaf) which is a 33 kd subunit. (2) Cytochrome b563 with two hemes, is a 23kd subunit. (3) a 20 kd (Fe-S) protein with (4Fe-4S) centers and (4) 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

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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 + H⎯→⎯ 2O 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.

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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 Edit ion, 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 edit ion. 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

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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., Balt imore, D., Berk, A. , Zipursky, S.L. , Metasudaira, P. and Darnell , J . 1995. Molecular cell Biology, 3r d Edition, Scienti f ic 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.

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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

Photosystem I

Light

P680*

P700*

Light

P700*

P680*

Proton gradient

O2 evolving complex

Plastocyanin

Cyt b6f

complex

−e

H2O

1/2O2

A0

A1

Fe-S

Fd

Fd:NADP+ oxidoreductase

NADP+

NADP+

Pheo

PQA

PQB

–1.0

1.0

0

PQA = plastoquinone PQB = second quinone A0 = electron acceptor chlorophyll A1 = phylloquinone

E’0

(vol

ts)

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 Edit ion, 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+.

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P680 P680* P680⎯⎯⎯ →⎯photon ⎯→⎯ps + + . −e 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, and two protons from the stroma to form plastoquinol, QH

BQ•

2 (Figure 10)

Q + 2e HQQe

H2⎯⎯⎯⎯ →⎯⎯→⎯++−•−

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.

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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 Edit ion, 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 O⎯→⎯ 2 + 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

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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. 4

H2

e3

e2

H

e1

H

e0 SSSSS

+

−−

+

−

+

−⎯→⎯⎯→⎯⎯→⎯⎯→⎯

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+ + ½O⎯→⎯ 2 + NADPH + H+

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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. ADP + Pi ATP + H

gradientproton

SynthaseATP ⎯⎯⎯⎯⎯⎯ → 2O

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. During the splitting of water by OEC of PSII, when 4 protons per O2 molecule formed are

released into the lumen of the thylakoid. 2. 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. 5t h Edit ion W.H. freeman and company, New York) 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, ∆G⎯→⎯ 0’ = 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 e- that 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 Edit ion, 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 x-ray 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 ry Neil Patterson Publishers. N. Carolena, USA) Whereas the elec photosystems is p

me electrons also pass in a linear flow to Ferridoxin and then to NAD and H2S is the source

4) ynthase.

as Rhodobacter capsulata and Rhodobacter mbrane protein (100kd) containing 4 polypeptide subunits and of the bacteriochlorphylls, pair of bacteriopheophytins, two

tran b

chrome bc1 oxidoreductase and mobile cytochrome C2. The pathway of s very similar to PSII of higher plants, algae and cyanobacteria [Figure 16(a)].

t

tron flow in higher plants, algae and cyacobacteria having tworedominantly linear, it is predominantly cyclic in bacteria. However, in green sulfur bacteria,

+soof 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 (bacterial ATP s

Photosynthesis in purple bacteria

he reaction center of purple bacteria suchTsphaeroides is a large integral me1-3 prosthetie groups, two pairs 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 smem rane α-helix. 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 cytoelectron flow iAbsorption 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.

Figure 16: The flow ugh the photosynthetic modules of – (a) purple

photosynthetic bacteria and (b) green sulfur bacteria lson, D.L. and CoxM (2005) Lehninger Principles of Biochemis ry. 4 t h

The ts electron to the posit form P870. Thus,

ght energy is converted and stored into chemical energy in the form of proton gradient which

1 rome C6 and bacterial ATP synthase but atic reaction [Figure 16(b)]. The excitation

ms P840+ radical. The electron

of electron thro

(Source: Ne tEdit ion, W.H. Freeman and company. New York)

reduced cytochrome C2 diffuses laterally to bacterial reaction center where it transfers iively charged special pair of reaction center (P870+) to

lidrives the synthesis of ATP by bacterial ATP synthase.

Photosynthesis in green sulfur bacteria

t involves Reaction center, Cytochrome bc and cytochIthe process is different and involves additional enzymof reaction center P840 to P840* releases an electron and forflows 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

P870* P840*

RC P840

Cyt bC1 mplex

RC P870

coExcitons

Excitons

Proton gradient

Pd-NAD reductase

Cyt bC1

complex

Proton gradient

Cyt C553

Cyt C2

Fe NAD—

NADP+

e—

Q

Pheo

e—

Q – 0.5

0

0.5

E’0

(vol

ts)

Purple bac(p in-quin

(a)

Green sulfur bacteria (FE-S type)

(b)

1.0

teria one type)

heophyt

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: H2S + H+ + NAD+ ⎯⎯ →⎯light 2H+ + S + NADH 12H2S + 6CO2 ⎯⎯ →⎯light C6H12O6 + 12S + 6H2O S ther organic compound as an electron source for the ome phososynthetic bacteria use H2 or o

ght de enden 2 (Tabl

Purple and green bacteria have only one photosystem. They use H2S or H2 i.e. compounds with e NAD+ to NADH.

he two Photosystem organisms use water as an electron donor where the reactions are:

H O 2H+ + 2e- + 1/2 O [E = + 0.815V], and

ons f ufficient to reach the

to NAD(P)+. The former is ngle photon absorbed by one PS in bacteria.

used to stabilize the earlier reactive ence, both PSII and PSI are coupled functionally and each acts linearly to boost

s mentioned earlier, the synthesis of carbohydrates [CO2 fixation] occurs in the stroma in a s, called the dark reactions as they do not require sunlight, although some of the lerated in the presence of light.

1946 to 1953. Calvin showed that the first roduct of CO2 fixation is 3-Phophogycerate by using 14CO2. These reactions are named after

d during this reaction, catalyzed te carboxylase / oxygenase, Rubisco, in stroma. Further

studies by Calvin’s group showed that 3-phosphoglycerate is converted back to ribulose

li p t reduction of CO e 1).

Why are there two photosystems in higher plants, algae and cyanobacteria?

lower (more negative) reduction potential as a source of electrons and reducThe reaction is H2S ⎯→⎯ S + 2H+ + 2e- [E0 = -0.25V] T ⎯→⎯2 2 0 NAD+ (P)+ + H+ + 2e- NAD (P) H-; [E0 = -0.32] Thus, less energy is required to boost electr rom H2S to a level sreduction potential of NAD+ than that to boost electrons from H2Opossible by a si 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 intermediates. Helectrons part of the way to reduce NADP+.

Dark reactions

Aseries of reactionreactions are acce Melvin Calvin, Andrew Benson and James A. Bassham had worked out the sequence of the dark reactions in growing cultures of algae betweenphim. 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 incorporateby the enzyme Ribulose 1,5bisphospha

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.

in addition uses several enzymes which make the Reductive Pentose cycle

n animals.

he enzymes involved are given in Figure 17 and Figure 8. Calvin cycle is discussed in 3 phases:

ibulose 1,5 bis phosphate (RuBP) to give 2 olecules of 3-Phosphoglycerate by the enzyme Ribulose 1,5 bisphosphate

his is the most abundant protein in plants, and probably on loroplast protein (1 of Figure 17).

to form 1,3 bisphosphoglycerate. The ,3 bisphosphogylcreate is reduced by NADPH to form glyceraldehydes 3- phosphate (2 and 3 of

important intermediate which can (a) be exose, starch, sucrose and (ii) Ribulose 1,5 bis

hus, fixation of 6 molecules of CO2 forms 12 molecules of Glyceraldehyde -3-Phosphate (G-3-

e pathway splits to make 1 molecule of Hexose and to regenerate the acceptor

7) as follows:

Calvin 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 i

Calvin cycle

The reactions of the Calvin cycle and t1

Phase I: CO2 Fixation

Carbon dioxide (CO2) is added to an acceptor, R(3-PG)m

carboxylase/oxygenses or Rubisco. Tearth, about 15% of ch

Phase II (a) : Reduction of 3 Phosphoglycerate

The 3-Phosophogylcerate is phosphorylated by ATP 1Figure 17). The Glyceraldehyde 3-phosphate is anconverted by a series of enzymes to form (i) Hphosphate 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. TP) and requires 12 ATP and 12 NADPH. At this stage, thmolecule, 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 1

28

(i) One glyceraldahyde-3-phosphate is converted to dihydroxyacctone phosphate (DHAP)

(v)

Phas

6-Ribulose 1, 2 2 osphate carboxylase).

ycerate + 12NADPH + 12H+ 12 glyceraldehyde 3-phosphate (NADP-enase).

acetone phosphate 3 fructose 1, 6-diphosphate

2

2 ribose 5-pho

thesis

by Triose phosphate isomerase. ( i i) One molecule of glyceraldhyde-3-phosphate and one of dihydroxyacetone

phosphate are condensed to form fructose1-6 diphosphate by the enzyme aldolase. Fructose 1-6 diphosphate is hydr(i i i) olysed by fructose diphosphatase in an irreversible reaction to give fructose-6-phosphate which is converted by a series of enzyme 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 enzyme Phosphoglucoisomerase forms Glucose-6- phosphate. Glucose-6-phosphate is converted by phosphoglucomutase to Glucose-1- phosphate which is the precursor of (a) disaccharide, sucrose , synthesized in the cytosol and (b) polysaccharide, 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.

e I Carboxylation. 5-diphosphate + 6CO + 6H O 12 3-phosphoglycerate (enzyme; ribulosc diph1

Phase II Reduction of phosphoglycerate 2 12 3-Phosphoglycerate + 12ATP 12 1,3-diphosphoglycerate + 12ADP (phosphoglycerate kinase)

12 1,3-diphosphogl3glyceraldehyde 3-phosphate dehydrog

Phase III Regeneration of CO2 acceptor 4 5 Glyceraldehyde 3-phosphate 5 dihydroxyacetone phosphate (triose phosphate isomerase)

3 Glyceraldehyde 3-phosphate + 3 dihydroxy5(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 4-

phosphate (transketolase). 8 2 Erythrose 4-phosphate + 2 dihydroxyacetone phosphate 2 sedoheptulose 1, 7-diphosphate

(aldolase) 9 2 Sedoheptulose 1, 7-diphosphate + 2H O 2 sedoheptulose 7-phosphate + 2Pi (sedoheptulose

diphosphatase) 10 2 Sedoheptulose 7-phosphate + 2 glyceraldehyde 3-phosphate sphate + 2 xylulose 5-

phosphate (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 paren

29

Figure 18: Calvin cycle depicting the CO2 ixation, Reduction and Regeneration phases which occur in the stroma and its relationsh with the sucrose synthesis in the cytosol

ource : Lodish, H., Balt imore, D., Berk, A. , Zipursky, S.L. , Metasudaira, P. and arnell , J . 1995. Molecular cell Biology, 3r d Edition, Scienti f ic American Books W.H.

freeman and company, USA)

fip

(SD

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 -3-

hosphate,

ted to Dihydroxyacetonephosphate. 2.

, 2 molecules of glyceraldehyde-3-phosphate and 2 molecules of Fructose -6- e

ate.

lase.

6. f glyceraldehydes-3-

9. 5Phosphate is then phosphorylated by ATP dependent

Thus, for the synthesis of 1 Hexose from 6CO , 12 molecule of NADPH and 18 molecules of A r

+ 12 NADPH + 12H2O C6H12O6 + 18 ADP + 18Pi + 12NADP+ + 6H+

tarch and sucrose are the major storage sugars in plants.

) Synthesis of starch

idues but is less branched than the animal storage sugar, the activated precursor ADP-glucose

the stroma.

phosphate (Stage IIb of Calvin cycle) which

e glucose unit of ADP-glucose is then transferred to the polysaccharide chain, to

P

1. Four molecules are converThere after, two molecules each of glyceraldehyde-3-phophate and Dihydroxy acctone phophate form 2 molecules of Fructose--6 phosphate by aldolase.

3. Thenphosphate form 2 xylulose -5- phosphate and 2 of erythrose-4- phosphate by the enzymtransketolase which requires the coenzyme thiamin pyrophosph

4. Two molecules erythrose-4-phosphate and two of dihydroxyacetonephosphate are converted to 2 molecule of seduheptulose 1,7 bis phosphate by aldo

5. 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 ophosphate form 2 molecules of xylulose-5-phosphate and 2 molecules of ribose-5-phosphate by Transketolase.

7. Four molecules of xylulose-5-phosphate are converted to four of Ribulose-5-phosphate by ribulose epimerase

8. Two molecules of ribose-5-phosphate are converted to 2 molecules of ribulose-5-phosphate by isomerase reaction. Six molecules of ribulose-phosphoribulo kinase to form Ribulose-1-5 bis phosphate.

2TP a e required in the Calvin Cycle and the net reaction of the Calvin cycle is.

6 CO2 + 18 ATP ⎯→⎯

Bio Synthesis of Starch and Sucrose

S

(a

Starch is a polymer of glucose resglycogen. It is stored in chloroplasts and synthesized fromin Fructose 6-phosphate is converted to Glucose-1-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. Thform starch.

31

ATP + glucose-1-phosphate ⎯→⎯ ADP-glucose + PPi

PPi ⎯→⎯ 2Pi

ADP-glucose + (glucose)n ⎯→⎯ (glucose

ucrose, a disaccharide, is synthesized in the cytosol. Hexose phosphates cannot be transported across the chloroplast trios rter transports triose

hosphate across the in ast to the cytosol for

es sucrose synthetase. Sucrose-6-phosphate is hydrolysed ure 18) which is exported to other parts of the plant through the veins.

omprises e is

d at the rate of about 4x109 ton/y which fixes about 1011 tons of CO2/y.

co has a low catalytic efficiency, k cat of 3 s . The carboxylation reaction bisco is shown in (Figure 19).

enolization to form Enediol Intermediate. The CO2 reacts ol Intermediate to from 2 carboxy-3-Ketoarabinitol 1,5 bisphosphate (CKAB).

ermediate. The stereospecific protonation of the ediate gives the second molecule of 3PG.

)n+1 + ADP

(b) Synthesis of Sucrose

S membranes. However, a ephosphate-Pi antiponer membrane of the chloropl an obligatory exchange p

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 enzymto sucrose (Fig

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 clmost 50% of the leaf protein and is the most abundant protein in the biosphere. This enzyma

estimated to be synthesize 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 Rubis -1

catalyzed by Ru The slow step of the reaction is thewith the EnediWater then reacts with 3-keto carbon, to form an adduct which cleaves to give a molecule of 3-phosphoglycerate (3 PG) and a carbanion intcarbanion interm

32

Figure 19: Carboxylase activity of Rubisco

Rubisco is inhibited by i yara binitol-1-phosphate AIP) and 2 carboxy-arabinitol-1,5, bisphosphate (CABP), the competitive inhibitors.

ts β-keto acid intermediate analogs, 2-carbox

(C

33

−232OPOCH −2

32OPOCH −−− −−−

his enzyme can a d in a later section. .2.3 Photorespiration)

egulation of Calvin cy

catalyzing the rate limiting step and (ii) other critical enzymes of the

2nd activation state of Rubisco.

uring the light reactions leads to an increase in the pH of stroma to 7.5 to

urn binds to CO2, thus activating the enzyme. s activation by CO2 and Mg results in the increase in maximum rate (Vmax) of

T lso catalyse an oxygenase reaction which will be describe(2

R cle

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)

ubiso, the first enzymeRCalvin cycle.

Regulation of Rubisco

Rubisco is activated by pH, CO2 and Mg2+. During photosynthesis, the rate of CO2 uptake is determined by availability of CO at the site of Carboxylation, the supply of Ribulose 1,5

isphosphate ab Pumping of protons d8.0 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 t

2P(Figure 20) Thithe 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 Seduheptulose-,7bis phosphatase, and Phosphoribulokinase are activated by reduced thioredoxin by sulfhydryl 1

exchange reaction. Thioredoxin is a small protein which has can be easily oxidized to form disulfide [S

two SH groups. These -SH groups -S-] bond. Thioredoxin is in turn activated by reduced

hen the chloroplasts are strongly illuminated, the NADP is converted to NADPH. Thus, NADP+ is not available for ferridoxin-ferridoxin by the enzyme ferridoxin-thioredoxin reductase. W

+

2

OHCH −−

COCHO 2

OHCH

COCHO

−−

OHCH −−

OHCH2

OHCH −−−2

32OPOCH

2-Carboxyarabinitol- 1-phosphate (CA1P)

2-Carboxyarabinitol- 1-5-biophosphate (CABP)

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-6-phosphate 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-6-Phosphate respectively are inhibited by reduced thioredoxin formed in presence of sunlight.

Figure (b) phosphatase and sedoheptulose 1,7 bis phosphatase

. Regulation also occurs by change in the ionization state of effector molecule, phosphoglycerate (PG) for phosphoribulokinase, PG inhibits the

inization of stroma (pumping out of proton) vours PG formation leading to the reversal of inhibition by PG2-.

20: Activation of Rubisco

Also, activities of fructose 1,6 bisare dependent on pH and Mg2+

2-

phosphoribulose kinase, but PG3- does not. Alkal3-fa

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-3-phosphate 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

ubisco is a bifunctional e associated oxygenase activity It catalyzes the oxygenation of Ribulose 1,5 bisphosphate (RuBP) at catalytic site on the L subunit as

e classical competitive inhibitors. Both the enzyme activities are inhib bisphosphate, CABP. The Km for CO2 is 12µM as against 250 ch higher than that for CO2. When O2 c 2 with Ribulose 1,5 bis phosphate to form ycerate in the chloroplast (Figure 22) Phosphoglycolate is toxic to plants being a powerful inhibitor of Triosephosphate Isomerase (an important enzyme ed to glycolate by a phosphoglycolate phosphatase. The oxygenase activity and the glycolate salvage pathway consuming O2 and releasing CO 2 cycle and occurs in three cellular compartments-chloroplasts

evolution when the hotosynthesis 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

R nzyme and has an the same

the carboxylation where carbondioxide and oxygen are thited by 2-carboxyarabinitol 1,5,

µM for O2 i.e. Km for O2 is mu

oncentration is high, Rubisco catalyzes the reaction of O phosphogycolate and 3-phosphogl

of glycolysis and Calvin cycle). It is dephosphorylat

2 is called Photorespiration or C, 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

igure 23). Calvin cycle (F 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 emperatures, low COt

p2 and high O2 concentrations. Early during

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

atmo No

Rubisco has been found that ha ation suggests that when CO2 concentratio sual to form ATP and NADPH which accum actions due to low availability of in the reduced electron carriers, proton gradient across the thylakoid m s,

ing ATP and NADP reaction to continue i.e. help in dissipating th ts damage to the photosynthetic components.

nation of leaves or chloroplasts in the absence of bot versible loss of their photosynthetic ability. SomePATHWAY which conserve CO late etc.) This is different from The C4 and CAM pathways are tw intaining photosynthesis on hot, dry days.

C4 Pathway

ists who elucidated this

millet - the agricultuhigh temp

Mesophyll cells As discussed in earlier section, CO esophyll cells.

lized bundle sheath cells lying below the me atmospheric oxygen (Figure 24). The bundle sheath cells contain carboxylase enzyme ts than those of C3

I which carries out cyclic electron flow, and therefore no O

he meseophyll cells are exposed to the outside, do not contain Rubisco but have

2s

sphere increased, photorespiration assumed significance. Inspite of its deleterious effects, why has the oxygenase activity of Rubisco been retained over the years during evolution?

s no oxygenase activity. One explann is low, but the sunlight is sufficient, the light reaction proceeds as u

ulate, as they are not utilized in the Calvin cycle re CO2. This, in turn, leads to an increase

embrane and photochemical excited states of the phostosystemwhich have deleterious effects on the Photosynthetic assemblies. Photorespiration by consum

H, provides ADP and NADP+ for the activity of the lighte photochemical energy and thus preven

This hypothesis is supported by the observation that bright illumih CO2 and O2 results in the rapid and irre

plants have bypassed the harmful effects of photorespiration by using the C4 and CAM 2 as C4 intermediates (oxaloacetate, ma

the Calvin cycle or the C3 pathway which uses C3 intermediates.

o evolutionary solutions for ma

C4 pathway is also known as Hatch-Slack pathway after the scientpathway in 1960s. The C4 cycle operates in several thousand species such as sugarcane, corn,

rally important plants and in tropical plants exposed to intense sunlight and eratures and dryness.

In C4 plants, there are two distinct types of photosynthetic cells (1) Bundle Sheath cells and (2)

2 fixation in the C3 plants occurs in the mHowever in C4 plants, the Calvin cycle photosynthesis occurs in specia

sophyll cells in the interior of the leaf and are protected from

chloroplasts and Rubisco but no phosphoenol pyruvate (PEP) . It has less grana and more starch in their chloroplas

plants and are unusual as they lack PSII and contain only PS2 is evolved.

Tphosphoenolpyruvate (PEP) carboxylase which captures CO2. The C4 pathway physically separates the CO capture from the Calvin cycle reactions in C4 plants. The C4 pathway is hown in Figure 25.

39

Figure 24: Diagram of the structure of leaves of C4 and C3 plants

olecular cell Biology, 3r d Edition, Scienti f ic American Books W.H. reeman and company, USA)

(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., Balt imore, D., Berk, A. , Zipursky, S.L. , Metasudaira, P. and Darnell , J . 1995. Mf

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

4 plants flourish in hot, dry, tropical climate whereas C plants predominate in temperate climate (low tC 3

emperature).

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, 1-9.

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 mak oxygen. Sci. Am., e 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 I-Photo 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.

6. ibid 1991. Regulatory phos1 phorylation of chloroplast antenna proteins. Trends Biochem. Sci. 16, 82-83.

Architecture of chloroplast coupling factor. Trends 17. McCarty, R.E., and Hammes, G.G. (1987). Molecular 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, 189-216.

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.

. and Maller, B.L. 1990. Photosystem I polypeptides. Physiologia Plan23. Scheller, H.V tarum, 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.