Photophosphorylation

Definition

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

• Photophosphorylation occurs in chloroplast.

• 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 ---------> O2 + (CH2O)

• Photosynthesis is divided into two segments

– (i) The light reactions / photophosphorylation

– (ii) The dark reactions, carbon-assimilation or carbon-fixation reactions:

• 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

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

• 2H2O+2NADP+ ------> 2NADPH+2H++2O2

• 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).

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

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).

• 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.

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 ---> an e- is excited from ground state to an excited (higher E) state

• excited molecule ---> ground state molecule + emits light or generates heat or does chemical work.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

PSI & PSII

• Photosystem I, PSI– reaction center designated by P700– chlorophyll a > chlorophyll b

• Photosystem II, PSII– reaction center designated by P680– contains equal amount of chlorophyll a and b.

• 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+.

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-).

• 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.

• 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).

• Overall reaction of PSII initiated by light:

• 4 P680 + 4H+ + 2PQB + 4 photons -------> (light)

4 P680+ + 2PQBH2

• H+ => from splitting of solvent water

• photons => from excited antenna molecules

• P680 ---> Pheo- ---> PQA ---> PQB ---> PQBH2 ---> diffuses away carrying its chemical energy to cytochrome bf complex ---> PSI

• Electrons in PQBH2 are transferred to cytochrome bf complex then to PSI.

• 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 ---------> 4H+ + 4e- + O2

• Four photons are required to break the bonds in water.

• 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 -------> 4 P680 + 4Z+

• 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-complex]0 -------->

• 4Z + [Mn-complex]4+

• Now, Mn complex can take 4 electrons from a pair of H2O.

• [Mn complex]4+ + 2H2O ------->

• [Mn complex]0 + 4H+ + O2

• 4H+ ==> released inside thylakoid lumen

• Fig. 19-51 Lehninger

• 2H2O ---------> 4H+ + 4e- + O2

• 4 P680+ + 4Z -------> 4 P680 + 4Z+

• 4Z+ + [Mn-complex]0 --------> • 4Z + [Mn-complex]4+

• [Mn complex]4+ + 2H2O ------->

• [Mn complex]0 + 4H+ + O2

• Sum of the above reactions:

• 2H2O + 2PQB + 4photons -----> O2 + 2QBH2

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+.

• 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+.

• 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+.

• Fig. 19-46 Lehninger

• P700* -----> A0 (e- acceptor) -----> A1

(phylloquinone) -----> Fe-S -----> Fd (ferridoxin) -----> ferridoxin-NADP+ oxidoreductase -----> NADP+

• 2Fdred + 2H+ + NADP+ -----> 2Fdox + NADPH + H+

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

• 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 ----> cytochrome c

• In chloroplasts: PQBH2 ----> plastocyanin

– Q cycle is involved– pumping of H+ across the membrane.

• 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.

• 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.

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

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

• 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.

• 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.

• 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.

• 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

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.

• 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).

• 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.

• Overall equation:

light

• ADP + Pi -------------> ATP + H2O

Ox. Phos. Vs. photophos.

Ox. Phos. Photophos.

Organelle mitochondria Chloroplast

Electrondonor

NADH H2O

Eo’ of half-rxn

-0.32 V 0.82 V

e- acceptor O2 NADP+

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