PHOTOSYNTHESIS: HARVESTING LIGHT ENERGY• The capture of solar energy by photosynthetic organisms and its conversion to the chemical energy of reduced organic compounds.• The overall equation for photosynthesis in vascular plants describes an oxidation-reduction reaction in which H2O donates electrons (as hydrogen) for the reduction of CO2 to carbohydrate (CH2O) CO2 + H2O O2 + (CH2O)
photosynthesis introduction Photosynthesis in plants encompasses two processes• The light-dependent reactions, or light reactions, which occur only when plants are illuminated, and the• carbon-assimilation reactions (or carbonfixation reactions), sometimes misleadingly called the dark reactions, which are driven by products of the light reactions• In the light reactions, chlorophyll and other pigments of photosynthetic cells absorb light energy and conserve it as ATP and NADPH; simultaneously, O2 is evolved. In the carbon-assimilation reactions, ATP and NADPH are used to reduce CO2 to form triose phosphates, starch, and sucrose, and other products derived from them. In this chapter we are concerned only with the light-dependent reactions that lead to the synthesis of ATP and NADPH.
Photosynthesis in plants takes place in Chloroplasts• Variable in shape• Few micrometers in diameter• Double membrane• Outer membrane is permeable to ions and small molecules• Inner membrane encloses the internal compartment :
• Thylakoids : Flattened , membrane surrounded vesicals• Grana: Thylakoids arranged in stacks• Stroma : Aqueous phase enclosed by inner membrane• Lumen : Phase enclosed by thylakoid membrane
Light drives electron Flow in Chloroplasts Robert hill in 1937 found• illumination of a leaf extract supplemented with dye became colourless and O2 was evolved• In the dark neither O2 evolution nor dye reduction• CO2 was neither required nor reduced• Hill reaction :Severo Ochoa found NADP+ as biological electron acceptor in chloroplasts•
Light Absorption• Visible light : electromagnetic radiation of wavelengths ranging from violet to red• Photon : Quantum of light• Energy of single photon of visible light is given by planck’s equation : E= and we know c = v lambda• Energy of a photon of visible light ranges from 150 kj/einstein (redlight) to 300kj/einstein (violet )• Energy is greater at violet end
• Quantum: The energy of an electron bound to an atom (at rest) is said to be quantized, which results in the stability of atoms, and of matter in general.• Excited state : A molecule that has absorbed a photon• Ground State : Excited molecule decays to the stable ground state giving up the absorbed quantum as light or heat or .....• Exciton: Quantum of energy passed from an excited molecule to another molecule• Exciton transfer : Process of transfer of exciton
Phtosynthetic pigments• Chlorophyll• Accessory pigments (carotenoids) 1. B(beta)-carotene 2. Lutein• Phycobilins 1. phycoeryhtrobilin (red in colour) 2. phycocyanobilin (blue in colour)Properties of phycobilins:• Open chain Tetrapyrroles• Extended polyene system• Dont have central Mg2+• Covalently linked to phycobiliproteins
Photosynthetic complexes• Light harvesting complex - chlorophyll molecules are fixed in relation to each other , to other protein complexes and to the membrane . - LHC II contains 7 molecules of chlorophyll a 5 of chlorophyll b 2 accessory pigments (lutein)• Phycobilisomes - cyanobacteria and red algae - highly ordered complexes - phycobilins covalently linked to the phycobiliproteins
Chlorophylls absorb light energy for photosynthesis• The most important light absorbing pigment• Green pigments• Ploycyclic planar structures• Long phytol side chain
Chlorophylls absorb light energy for photosynthesis• Amount of chlorophyll a to chlorophyll b in most chloroplasts is twice• Types of chlorophyll i. Chlorophyll a ii. Chlorophyll b iii. Chlorophyll c1 iv. Chlorophyll c2 v. Chlorophyll d vi. Chlorophyll f
Difference b/w haemoglobin and chlorophyll protopryphyrin ring• Has Mg2+ instead of iron in the central postion of protoporyphyrin ring as compared to the haemoglobin• Chlorophylls have a fifth five memberd ring not present in heme .
Accessory pigments• Carotenoids absorb wavelength that is not absorbed by the chlorophyll• Extend the range of light absorption• B carotene - red orange isoprenoid (5 carbon structure) - poly ene• Lutein - yellow
Phycobilins• linear tertapyrroles• structurally related to chlorophyll a But lack the phytol side chain & magnesium ion• They claim a unique ecological niche - in red algae and in cyanobacteria absorb light in range 520 – 630 nm -occupy the niches where light of lower and higher wavelength is filtered out .
Organization of photosystems in the thylakoid membrane.The light-absorbing pigments of thylakoid or bacterial membranesare arranged in functional arrays called photosystems • Photosystems are tightly packed in the thylakoid membrane, with several hundred antenna chlorophylls and accessory pigments surrounding a photoreaction center. • Absorption of a photon by any of the antenna chlorophylls leads to excitation of the reaction center by exciton transfer (black arrow). Also embedded in the thylakoid membrane are the cytochrome b6f complex and ATP synthase
How does the Chlorophyll funnels the energy to reaction centers by exciton transfer???? • The excited antenna chlorophyll transfers energy directly to a neighboring chlorophyll molecule, which becomes excited as the first molecule returns to its ground state • very little fluorescence is observed •This transfer of energy, exciton transfer, extends to a third,fourth, or subsequent neighbor, until one of a special pair of chlorophyll a molecules at the photochemical reaction center is excited
• In this excited chlorophyll molecule, an electron ispromoted to a higher energy orbital. This electronthen passes to a nearby electron acceptor that ispart of the electron-transfer chain, leaving thereaction-center chlorophyll with a missing electron(an “electron hole,” denoted by + in fig )• The electron acceptor acquires a negative charge inthis transaction. The electron lost by the reaction-center chlorophyll is replaced by an electron from aneighboring electron-donor molecule (step 5 ),which thereby becomes positively charged. In thisway, excitation by light causes electric chargeseparation and initiates an oxidation-reductionchain.
Absorption spectra of the photosynthetic pigments• Absorption spectrum – a plot of the amount of light absorbed across a series of wavelengths while• Action spectrum – a measure of the rate of photosynthesis as a function of wavelengths of light
Absorption spectra of the photosynthetic pigments• 3 classes of photosynthetic pigment absorb light of different wavelengths – chlorophylls – absorb in the blue (450nm) and red (650-700nm) regions of the visible spectrum – carotenoids – absorb in the range of 400-500 nm – phycobilins – absorb in the range of 500-650 nm
Stages of Photosynthesis• A two stage process; – The Light Dependent Process (Light Reactions) • occur in the grana – The Light Independent Process (or Dark Reactions) • take place in the stroma of the chloroplasts• Recent evidence suggests that a major enzyme of the Dark Reaction is indirectly stimulated by light, thus the term Dark Reaction is somewhat of a misnomer.
Light phase of photosynthesis• involves the use of light energy to generate ATP & reducing power – reducing power in photosynthetic organisms of plants is NADPH• e- required to convert NADP+ into NADPH are derived from H2O• O2, the oxidized component of redox system ½O2/H2O is generated as a by- product• The light reactions of photosynthesis involve the following • Capture of a photon of light by a chlorophyll photosystem • Conversion of photon energy to energy stored by electrons (chemical energy) • Capture of this chemical energy by the reaction center chlorophyll of a photosystem • Oxidation of the reaction center chlorophyll by the primary electron acceptor
Photosystems• Two photosystems act in tandem in light reactions• each has its own type of photochemical reaction center and set of antenna molecules• Two photosystems are Photosystem II and Photosystem I• Photosystem II - equal amounts of chlorophylls a and b - consist of units each with one molecule of chlorophyll a (P680) & 250 antenna molecules - optimally absorbs photons of wavelength of 680 nm localized in the stacked(appressed) membrane regions - Excitation of its reaction center P-680 drives electrons through the cytochrome b6f complex with concomitant movement of protons across the thylakoid membrane
Photosystems• Photosystem I : - It has more chlorophyll a then b - Composed of units each with one molecule of chlorophyll a (P700), constituting the reaction centre & 250 antenna pigments - optimally absorbs photons of wavelength of 700 nm - localized in the unstacked region of thylakoid membrane - Excited p700 passes electrons to the Fe-S protein ferredoxin then to NADP+
Light reactions (photosynthesis)• Electrons b/w the two photosystems are carried by the soluble protein plastocyanin• Z scheme as the route of electrons from H20 to NADP+
Light reactions (photosynthesis)• For every two photons absorbed (one by each photosystem) , one electron is transferred from H2O to NADP+• So to form one molecule of O2 , which requires transfer of 4 electrons of two H2O number of photons absorbed should be eight (four by each photosystem)
Steps in PS II• Excitation of P680 in PSII produces P680*, an excellent electron donor that within picoseconds, transfers an electron to pheophytin, giving it a negative charge• With the loss of its electron, P680* is transformed into a radical cation, P680+• Pheo very rapidly passes its extra electron to a protein-bound plastoquinone, PQA (or QA), which in turn passes its electron to another, more loosely bound plastoquinone, PQB (or QB).
Steps in PS II• When PQB has acquired two electrons in two such transfers from PQA and two protons from the solvent water, it is in its fully reduced quinol form, PQBH2• Eventually, the electrons in PQBH2 pass through the cytochrome b6 f complex .• Binding site for plastoquinone is the point of action of many commercial herbicides that kill plants
Steps in PS I• Electrons are carried b/w two photosystems by a soluble protein plastocyanin• The excited reaction center P700* loses an electron to an acceptor, A0 creating A0- and P700+• again, excitation results in charge separation at the photochemical reaction center• P700 is a strong oxidizing agent, which quickly acquires an electron from plastocyanin, a soluble Cu-containing electron-transfer protein
• A0- is an exceptionally strong reducing agent that passes its electron through a chain of carriers that leads to NADP+.• First, phylloquinone (A1) accepts an electron and passes it to an iron- sulfur protein (through three Fe-S centers in PSI)• From here, the electron moves to ferredoxin (Fd), another iron-sulfur protein loosely associated with the thylakoid membrane• The fourth electron carrier in the chain is the flavoprotein ferredoxin which is an NADP oxidoreductase and transfers electrons from reduced ferredoxin (Fdred) to NADP+
Cytochrome b6f complex links photosystem II and I• Electrons due to excitation of p680 are carried to p700 via cytochrome b6f complex and plastocyanin• Complex contains b-type cytochrome(with two heme groups bH and bL ) , cytchrome f and a reiske iron sulfur protein• Function of this complex involves Q cycle in which electrons pass one at a time from PQBH2 to cytochrome b6• Cycle results in pumping of protons across the membrane• 4 protons moving for each pair of electrons• Create the potential gradient for the ATP synthesis (PH 8 of stroma and PH 4 of lumen )
Cyclic Electron Flow (cyclic photophosphorylation)• Involves only PS I, not PS II• Electrons passing from p700 to ferredoxin do not continue to NADP + but move back through the Cytochrome b6f complex to plastocyanin .• Electrons are repeatedly recycled
Cyclic Electron Flow (cyclic photophosphorylation)• Each electron is propelled by the energy of one photon• NADPH is not formed• O2 is not evolved• Proton pumping by Cytochrome b6f complex• Phosphorylation of ADP to ATP thats why its called cyclic photophosphorylation
ATP synthesis by photophosphorylation• Reaction centers, electron carriers and ATP forming enzymes are located on the proton impermeable membrane (thylakoid membrane)• ATP synthesis is catalyzed by Fo F1 complexes similar in structure and function to mitochondrial complexes• Electron transferring molecules in the chain are asymmetrically arranged results in net movement of protons from stromal side to the lumen• ATP SYNTHASE is the enzyme responsible for the production of ATP
ATP synthesis byphotophosphorylation
ATP SYNTHASE• Large complex• Two functional components , CFo and CF1• CFo is the transmembrane proton pore• CF1 is the peripheral membrane protein complex• ATP SYNTHASE complexes as knob like projections on outside• CF1 portion of ATP synthase is located on more alkaline side of membrane
Proton gradient in relation to ATP SYNTHASE orientation
Photophosphorylation VS Oxidative PhosphorylationPhotophosphorylation Oxidative Phosphorylation the chemiosmotic involve a chemiosmotic phosphorylation that makes ATP mechanism of ADP during photosynthesis phosphorylation the electrons that drive pumping hydrogen ion pumping is driven are donated by chlorophyll by electrons donated by NADH or FADH2 the energy is ultimately derived the energy is ultimately derived from photons from chemical bonds
State transitions change the distribution of LHC II b/w two photosystems• Why there was a need to separate the two photosystems ?????
State transitions change the distribution of LHC II b/w two photosystems• As because the two photosystems PSI and PSII were physically contiguous , excitons originating in the antenna system of PSII would migrate directly to the reaction center of PSI leaving the reaction center of PSII under excited so interfering with the operation of two center system• Lets discuss its detail
State transitions change the distribution of LHC II b/w two photosystems• LHC II is associated with PSI and PSII and this depends on the light intensity and wavelength• There are two state transitions associated with the LHC II• In State I , a serine residue in LHC II is not phosphorylated so LHC II associates with PSII• When light absorbed by PS II , photosystem reduces plastoquinone to plastoquinol( PQH2)
State transitions change the distribution of LHC II b/w two photosystems• PQH2 activates the protein kinase wich triggers the transition to state II by phosphorylating the Thr residue on LHC II• Phosphorylation results in the disssocition of LHCII from PSII to PSI so LHC II captures the electrons for PSI• PSI oxidizes PQH2 faster then the PSII would make it and so PQH2 is converted to PQ and PQ(plastoquinone ) triggers the dephopshorylation of Thr residue on LHC II
State transitions change the distribution of LHC II b/w two photosystems
Water is split by Oxygen Evolving Complex• Four electrons abstracted from water do not directly pass to P680+• P680+ can accept only one electron• Oxygen evolving complex passes four electrons one at a time to P680+• Immediate electron donor to P680+ is Tyr residue• Four photons are required to provide the energy for splitting• water splitting complex has a cluster of four magnesium ions (Mn complex)
Water is split by Oxygen Evolving Complex• When 2 P680* entities have each donated an e- to Q, they become P680*· cationic free radicals.• In order to keep the process going they must each receive an e- to reduce them to ground state P680.• The 2 e- which reduce P680+· to ground state, are derived from H2O• Oxygen is produced as a by-product
Water is split by Oxygen Evolving Complex• P680 + , a very strong oxidant, extracts e- from water molecules bound at the manganese center• The structure of this center, which includes 4 manganese ions, a calcium ion, a chloride ion, and a tyrosine residue that forms a radical, has not been fully established,
Water is split by Oxygen Evolving Complex• Manganese was apparently evolutionarily selected for this role – because of its ability to exist in multiple oxidation states (Mn+2, Mn+3, Mn+4, Mn+5) and to form strong bonds with oxygen-containing species• The manganese center, in its reduced form, oxidizes two molecules of water to form a single molecule of oxygen• The absorption of one photon results in the accumulation of one +ive charge on Mn
Water is split by Oxygen Evolving Complex
Water is split by Oxygen Evolving Complex• Each time the absorbance of a photon kicks an e- out of P680, the +ively charged special pair extracts an e- from the manganese center• Thus, 4 photochemical steps are required to extract the e- and reduce the manganese center• The four e- harvested from H2O are used to reduce 2 molecules of PQ to PQH2
Comparison of Non-cyclic & Cyclic Photophosphorylation
Stoichiometry of photophosphorylation• 12 H+ ions move from the stroma to the thylakoid lumen per four electrons passed• 4 of them are moved by Oxygen evolving complex• 8 of them are moved by Cytochrome b6f complex• This creates the proton gradient• one photon per electron at each photo reaction center• Eight photons must be absorbed to drive 4 electrons from H2O to NADP+