Photophosphorylation

1
Photophosphorylation
2
Definition

• Photophosphorylation is the process by which
  photosynthetic organisms use the energy of
  sunlight to produce ATP and NADPH.
• Photophosphorylation occurs in chloroplast.

3

• Fig. 19-34 Lehninger
• Photophosphorylation is the first step in
  photosynthesis, the light-dependent reduction of
  CO2 by H2O to make carbohydrates
  
  
  light
• CO2 H2O ---------gt O2 (CH2O)
  

4

• Photosynthesis is divided into two segments
• (i) The light reactions / photophosphorylation
• (ii) The dark reactions, carbon-assimilation or
  carbon-fixation reactions

5

• The light reactions / photophosphorylation
• occur only when plants are illuminated
• produces ATP and NADPH
• The dark reactions, carbon-assimilation or
  carbon-fixation reactions
• occur at all time (not just in the dark)
• depend on the light reactions
• uses ATP and NADPH to convert CO2 to glucose

6
Photophosphorylation

• Electron donor water (poor electron donor, large
  ve E0)
• Electrons are transferred from water to NADP,
  and the energy required is derived from sunlight.
  light
• 2H2O2NADP ------gt 2NADPH2H2O2

7

• Electrons flow through a series of membrane-bound
  carriers (cytochromes, quinones and Fe-S
  proteins).
• During electron flow, protons are pumped to
  create a proton gradient.
• This is used to drive ATP synthesis from ADP and
  Pi by ATP synthase (similar to oxidative
  phosphorylation).

8
Structure of Chloroplast

• Fig. 19-35 Lehninger
• Has two membranes
• outer membrane - permeable
• inner membrane - encloses inner compartment
• Inner compartment
• has membrane enclosed sacs called thylakoids

9
chloroplast

• Thylakoid membrane
• contains pigments (chlorophyll and carotenoid)
  of photophosphorylation and the enzymes for ATP
  synthesis.
• Stroma
• the aqueous compartment contained within the
  inner membrane
• site of carbon fixation (synthesis of
  carbohydrate - dark reaction).

10

• In ox-phos.
• electrons flow from NADH to O2
• In photophos.
• electrons flow from H2O to NADP
• Major difference
• In ox-phos. NADH is a strong electron donor.
• In photophos. H2O is a poor electron donor.

11
Light

• Visible light 400-700 nm of the spectrum
• An einstein (1 mol) of visible light 170 (red)
  and 300 (violet) kJ of energy
• molecule light ---gt an e- is excited from
  ground state to an excited (higher E) state
• excited molecule ---gt ground state molecule
  emits light or generates heat or does chemical
  work.

12

• Fig. 19-37 Lehninger
• Thylakoid membrane contains a number of pigments
  that can absorb the entire spectrum present in
  the sunlight.
• Chlorophylls - primary light-absorbing pigments.
• Carotenoids - accessory pigments.

13

• Fig. 19-42 Lehninger
• The light-absorbing pigments are arranged in
  units called photosystems.
• They might contain a few hundred-pigment
  molecules.
• All the pigments can absorb light but only a few
  chlorophylls are associated with the reaction
  center.
• Chlorophylls in the reaction center transduces
  light energy into chemical energy.

14

• The other pigments serve as light harvesting
  antenna molecules.
• They absorb light and funnel it to the reaction
  center by transferring the energy to adjacent
  pigments.
• An antenna molecule (chlorophyll or accessory
  pigment) is excited to higher energy level by
  absorbing light.

15

• Fig. 19-43 Lehninger
• The excited antenna molecule transfers its energy
  to a neighbouring chlorophyll molecule and
  returns to its ground state.
• This energy transfer is called exciton transfer
  (resonance energy transfer).
• This is repeated to 3rd, 4th, etc. until a
  chlorophyll molecule at the photochemical
  reaction center is excited.

16

• The energy is transferred to a reaction center
  chlorophyll, exciting it.
• The excited reaction center chlorophyll passes an
  electron to an electron acceptor.
• The reaction-center chlorophyll has an empty
  orbital (an electron hole).
• The electron acceptor acquires a negative charge.
  

17

• The electron hole in the reaction center is
  filled by an electron from a neighbouring
  electron donor molecule.
• The electron donor molecule becomes positively
  charged.
• The absorption of light causes electric charge
  separation in the reaction center and initiates
  an oxidation-reduction reaction.

18

• Photosystem set of light absorbing pigments.
• Plants (thylakoid membrane of chloroplasts) have
  two reaction centers
• Photosystem I, PSI
• Photosystem II, PSII
• Each photosystem has over 200 molecules of
  chlorophylls and about 50 molecules of
  carotenoids.

19
PSI PSII

• Photosystem I, PSI
• reaction center designated by P700
• chlorophyll a gt chlorophyll b
• Photosystem II, PSII
• reaction center designated by P680
• contains equal amount of chlorophyll a and b.

20

• PSI and PSII have distinct and complementary
  functions.
• PSI and PSII act in tandem (one after another) to
  catalyze the light driven movement of electrons
  from H2O to NADP.

21
Photosystem II, PSII

• P680, the chlorophyll in the reaction center of
  PSII, absorbs a photon of light.
• This promotes the electron to the excited stage.
• Excited reaction center P680, loses its electron
  to pheophytin (a chlorophyll like accessory
  pigment), giving it a negative charge (Pheo-).

22

• Tyr residue (represented as Z) on D1 protein of
  PSII, donates an electron to P680.
• Pheo- rapidly passes its electron to a
  protein-bound plastoquinone, PQA.
• PQA passes its electron to another plastoquinone,
  PQB.

23

• When PQB receives two electrons from PQA (in two
  transfers) and two protons from the solvent
  water, it is in quinol form, PQBH2 (fully reduced
  form).

24

• Overall reaction of PSII initiated by light
• 4 P680 4H 2PQB 4 photons -------gt
  (light)
  
  4 P680 2PQBH2
• H gt from splitting of solvent water
• photons gt from excited antenna molecules

25

• P680 ---gt Pheo- ---gt PQA ---gt PQB ---gt PQBH2 ---gt
  diffuses away carrying its chemical energy to
  cytochrome bf complex ---gt PSI
• Electrons in PQBH2 are transferred to cytochrome
  bf complex then to PSI.

26

• P680 must acquire an electron to return to its
  ground state to capture another photon energy.
• P680 acquires electron from the splitting of
  water.
• 2H2O ---------gt 4H 4e- O2
• Four photons are required to break the bonds in
  water.

27

• Water splitting Mn-complex passes four electrons
  one at a time to P680. (P680 can accept only
  one electron at a time).
• The immediate electron donor to P680 is a Tyr
  residue (designated as Z) in protein DI of PSII
  reaction center.
• 4 P680 4Z -------gt 4 P680 4Z

28

• Tyr (Z) regains its electron by oxidizing a
  cluster of 4 Mn ions in the water-splitting
  complex. Mn cluster becomes more oxidised.
• 4Z Mn-complex0 --------gt
• 4Z
  Mn-complex4

29

• Now, Mn complex can take 4 electrons from a pair
  of H2O.
• Mn complex4 2H2O -------gt
• Mn complex0 4H
  O2
• 4H gt released inside thylakoid lumen

30

• Fig. 19-51 Lehninger
• 2H2O ---------gt 4H 4e- O2
• 4 P680 4Z -------gt 4 P680 4Z
• 4Z Mn-complex0 --------gt
• 4Z
  Mn-complex4
• Mn complex4 2H2O -------gt
• Mn complex0 4H
  O2
• Sum of the above reactions
• 2H2O 2PQB 4photons -----gt O2 2QBH2

31
Photosystem I, PSI

• Photochemical events are similar to those in
  PSII.
• Light is absorbed by antenna molecules and the
  energy is transferred to P700 (reaction center)
  by resonance energy transfer.
• The excited reaction center P700 loses an
  electron to an electron acceptor, A0 (like
  pheophytin in PSII) creating A0- and P700.

32

• This results in charge separation at the
  photochemical reaction center.
• P700 is a strong oxidizing agent. It acquires an
  electron from plastocyanin, a soluble
  Cu-containing electron transfer protein.
• A0- is a strong reducing agent. It passes its
  electrons through a chain of carriers leading to
  NADP.

33

• A0- passes its electrons to phylloquinone, A1
• A1 passes it to an Fe-S protein
• Fe-S protein passes the electron to ferredoxin,
  Fd (another Fe-S protein).
• The electron is then transferred to a
  flavoprotein, ferredoxin-NADP oxidoreductase.
  The electron is transferred from reduced Fd to
  NADP.

34

• Fig. 19-46 Lehninger
• P700 -----gt A0 (e- acceptor) -----gt A1
  (phylloquinone) -----gt Fe-S -----gt Fd
  (ferridoxin) -----gt ferridoxin-NADP
  oxidoreductase -----gt NADP
• 2Fdred 2H NADP -----gt 2Fdox
  NADPH H

35
Cytochrome bf complex links PSII and PSI

• Electrons temporarily stored in Plastoquinol
  (PQBH2) in PSII are carried to PSI via the
  cytochrome bf complex and the soluble protein
  plastocyanin.
• Cyt bf complex contains
• cytochrome b (with two heme groups),
• Fe-S protein, and
• cytochrome f

36

• Fig. 19-49 Lehninger
• Cyt bf complex is like complex III of
  mitochondria.
• Cytochrome bf,
• transfers electrons from a mobile lipid soluble
  carrier to water soluble protein.
• In mitochondria UQH2 ----gt cytochrome c
• In chloroplasts PQBH2 ----gt plastocyanin
• Q cycle is involved
• pumping of H across the membrane.

37

• H moves from stroma to the thylakoid lumen. (4H
  move for each pair of electrons).
• Electron flow from PSII to PSI result in the
  production of H gradient across the thylakoid
  membrane.

38

• Volume of the flattened thylakoid lumen is small.
  
• Therefore, small H flux into lumen can create
  large pH difference between stroma (pH 8) and
  lumen (pH 5) - a powerful driving force for
  ATP synthesis.

39
ATP synthesis

• Fig. 19-52 Lehninger
• PSII and PSI
• electrons are transferred from water to NADP
• protons are pumped across the thylakoid membrane.
• proton gradient drives the synthesis of ATP from
  ADP and Pi

40
ATP synthase

• ATP synthase of chloroplast is like that of
  mitochondria.
• ATP synthase has two components
• CF0 - like F0 in mitochondria
• integral membrane protein
• a transmembrane proton pore
• CF1 - like F1 in mitochondria
• peripheral membrane protein
• binding site for ATP and ADP

41

• Fig. 19-53 Lehninger
• ATP synthase is on the outside surface (stroma
  side) of thylakoid membrane.
• ATP synthase is on the inside (matrix side) of
  inner mitochodrial membrane.
• H pumped into lumen through cyt bf and water
  splitting Mn complex and returned to outside via
  ATP synthase.
• H pumped out via complexes and returned to
  matrix via ATP synthase.

42

• Both orientation and the direction of H pumping
  in chloroplasts are opposite to those in
  mitochondria.
• In both cases, F1 portion of ATP synthase is
  located on the more alkaline side (N) of the
  membrane, and H flow down their concentration
  gradient.

43

• The mechanism of chloroplast ATP synthase is
  believed to be identical to that of mitochondria.
• ADP and Pi condense to form ATP on CF1 and the
  flow of H causes ATP to be released from CF1.

44

• Similarity between ox. phos. and photophos.
• electron transfer
• formation of proton gradient
• ATP synthase complex
• comparison of topology of H movement and ATP
  synthase orientation in mitochondria and
  chloroplasts
• ATP synthesis

45
Cyclic electron flow produces ATP but not NADPH
or O2

• At some point, plant cells do not require much
  NADPH for biosynthesis process but still require
  ATP for other biological process.
• It needs to vary the ratio of NADPH and ATP
  formed.
• This is done by an alternative path of light
  induced electron flow, called cyclic electron
  flow.

46

• Cyclic electron flow involves only PSI.
• Electrons passed from P700 to ferredoxin do not
  continue to NADP, but move back through the
  cytochrome bf complex to plastocyanin.
• Plastocyanin donates electrons to P700, which
  transfers them ferridoxin (in a cycle).

47

• No net formation of NADPH or splitting of H2O to
  O2.
• H is pumped by cytochrome bf complex (generation
  of H gradient) and ATP is synthesized.
• Cyclic electron flow and photophosphorylation
  together is known as cyclic photophosphorylation.

48

• Overall equation
  
  
  light
• ADP Pi -------------gt ATP H2O

49
Ox. Phos. Vs. photophos.