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 Stroma 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.: In photophos.: Major difference: 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 Photosystem II, PSII 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

Cyt bf complex is like complex III of mitochondria. Cytochrome bf, 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 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.