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MCB102-10 - MCB102 Photosynthesis Light Reactions Fall 2008...

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Unformatted text preview: MCB102 Photosynthesis Light Reactions Fall 2008 Reading: pages 742-764 Outline 1. Photosynthesis can split two water molecules, releasing oxygen, reducing power (4 electrons), 4 hydrogen ions, making 2 NADPH and about 3 ATP. 2. Light-dependent activities take place in the chloroplast's thylakoid membrane. 3. Light is absorbed by antenna pigments and is transferred to a photosynthetic reaction center. 4. In purple bacteria, light causes a pH gradient to form across the cell membrane, resulting in ATP synthesis. 5. In green sulfur bacteria light causes the synthesis of NADH and the oxidation of H28 to S. 6. Cyanobacteria and plants have two photosynthetic reaction centers, corresponding to those of purple bacteria and green sulfur bacteria. 7. One center splits water, making oxygen and transporting four electrons and four hydrogen ions. The hydrogen ion transport contributes to ATP synthesis. 8. An intermediate similar to complex III of oxidative phosphorylation causes transport of 2 hydrogen ions per electron, contributing to ATP synthesis. 9. The second photosynthetic reaction center reduces NADP+ to NADPH + H+ 10. Cyanobacteria and plants can make ATP Without making NADPH. 11. Antenna pigments that serve one photosynthetic reaction center can leave it and serve the other photosynthetic reaction center when necessary. Chemical Equations of Photosynthesis In 1780 Joseph Priestley depleted the air in a bell jar of oxygen by burning a candle until it went out. Then he showed that allowing a plant to grow inside the jar caused oxygen to be regenerated, so that the candle could be relit, and a mouse could breathe. Shortly thereafter, Jan Ingenhousz showed that light was necessary for the regeneration of oxygen by the plant. Jean Senebier demonstrated that carbon dioxide was taken up in the process of photosynthesis. He noted that plants under water release oxygen bubbles on their surface only when carbon dioxide is dissolved in the water. Theodore de Saussure showed that the weight of plants was more than the weight of carbon consumed and postulated that water must be taken up, as well as something from the air. In 1931 Cornelius Van Niel perceived a common pattern in the reactions of photosynthesis carried out by green plants and by green sulfur bacteria. Green sulfur bacteria oxidize hydrogen sulfide to elemental sulfur during photosynthesis, following the equation 002 + 2 H28 9 (CH20) + ZS + H20 Following this pattern, he said green plants must oxidize water to oxygen. 002 + 2 H20 ~> (CH20) + 02 + H20 When the heavy isotope of oxygen, 180, became available in 1941, Samuel Rubin and Martin Kamen at Lawrence Berkeley Laboratory showed that heavy water yields heavy oxygen during photosynthesis. Thus the oxygen comes from the water and not from the carbon dioxide. In 1937 Robert Hill showed that chloroplasts evolve oxygen when they are illuminated in the presence of an electron acceptor such as ferricyanide. Ferricyanide, which is blue, is reduced to ferrocyanide, which is colorless. 2H20+4Fe3+—)02+4H++4Fe2+ Thus oxygen can be released without fixing carbon. This part of photosynthesis is discussed in today's lecture. Since it depends upon light as a source of energy, it is called the light reactions. Light absorbing pigments Both plants and photosynthetic bacteria contain chlorophyll (meaning “yellow-green leaf’ in Greek), which is a cyclic tetrapyrrole, similar to hemoglobin, but with an extra 5-membered ring. Instead of containing iron, like hemoglobin, chlorophyll contains a magnesium ion. Richard Willstatter suggested that it plays a role in photosynthesis in 1915. Emil Fischer’s son Hans solved its structure in 1940. Only about one out of 2,500 chlorophyll molecules is in a reaction center that converts light energy into chemical energy. Most of the chlorophyll molecules act as antennae to absorb light and pass it on to other chlorophyll molecules and eventually to the photosynthetic reaction centers. The antenna chlorophylls are associated with special proteins that make up light harvesting complexes. Carotenes, which also absorb light, are found in light harvesting complexes. They absorb a shorter wavelength of light (with higher energy) than does chlorophyll. The carotenes pass this energy on to chlorophyll, because the absorption spectra of carotenes and chlorophyll overlap, as shown on the figure on the next page. When leaves die in the fall, chlorophyll is rapidly degraded, because its degradation intermediates are toxic. The carotenes remain and make the leaves orange or yellow. Carotenes can act as antioxidants. The second figure on the next page shows the conjugated systems of alternating single and double bonds in chlorophyll and carotene as shaded. These conjugated systems absorb photons. {mass Q swamp)th A . in, ,l 3 W _ §——-> ‘ 4" _._W.,.-,N_l,, “Wm l "‘70 \ 3 lCH CH J Saturated bond in g ? [Jr/“K lbactericchlomphyll? H '3*"CH2CH3 A WNW phy‘ml side chain CH3 h _ h.-,,,l.__.w.,,.-__. ,{fl CH3 CH3 CH3 ‘1? H , \8 \/ \//[\\//\// \/\/\\\//\O/ \CI/{g HV \(1 / \\ CH30 o Chlorophyll a CH3“ xCHg CH3 CH3 _ HgC/l/ k m CH CH3 CH3 CH3 3 6 Carotene Ram fig Nd” 2'00 The light reactions at the photosvnthetic reaction centers of bacteria Purple bacteria (Photosystem II, which was discovered second) In 1952 Louis Duysens in the Netherlands found that illumination of purple bacteria With light at 870 nm caused a decrease in absorption that was reversed after time in the dark. He called this effect “photo-bleaching” and proposed that the photosynthetic pigment loses an electron after absorption of light, and that the pigment gets the electron back in a cyclic process that creates chemical energy. Robert Huber’s group in Munich crystallized the membrane-bound photosynthetic reaction center of the purple bacterium Rhodopseudomonas viridas and determined its structure by X-ray diffraction analysis. Spectrscopy of Visible light and electron paramagnetic resonance have been used to determine the path of excited electrons. A special pair of chlorophylls, Whose maximum absorption wavelength is 870 nm, absorbs one photon. Thus this pair is called P870, and P stands for “pigment.” The absorption of the photon causes an electron to go to an excited state in a higher orbital. When excited, it has a reduction potential of about -1 V, as compared to the reduction potential of NADPH, which is about -0.3 V. Thus, there is plenty of energy trapped for making ATP or NADPH. The excited pair of chlorophylls is denoted P870*. The excited electron is passed to a pheophytin, Which is similar to chlorophyll, except that magnesium is replaced by two hydrogen ions. Thus the special chlorophyll pair is oxidized to P870+ and the pheophytin is reduced. The excited electron is then passed to one quinone and then another quinone. The second quinone, called QB, retains the electron until a second electron has reached it from the excitation caused by a second photon. QB then takes up two hydrogens from the cytosolic side of the cell membrane. The electrons are then passed to a cytochrome bcl complex, which is similar to complex III of mitochondria. When two electrons are passed from the cytochrome bcl complex to cytochrome cg, four hydrogen ions are 440 L transported to the outside of the cell membrane, creating a pH gradient that drives ATP synthase. Cytochrome 02 passes R electrons back to P87 0+ to restart the cycle. Purple bacteria “.05 in (pheophytin—quinone type) (a) HGURE 19—54 Functional modules of photosynthetic machinery in purple bacteria and green sulfur bacteria. (3) En purple bacteria, light energy drives electrons from {he reaciics~<:enter P870 through chec— E’” (volts) phytén {sheet a quinooe {Q}, and the cytochrome ac. Complex, than through cytochrome C2 and thus back to the reaction center. Electron é through the cytochron‘ec in} complex causes proton pumping, cre< eéectrochemicai poacméal the: powers A??? synthesis. fisgé 4 X Prater} gradient Green sulfur bacteria. (Photosystem I, which was discovered first) In green sulfur bacteria, a chlorophyll molecule that absorbs at 840 nm absorbs a photon. An electron is excited, and it can go to a quinone and through a cytochrome be; complex, which pumps two hydrogen ions across the cell membrane and makes a proton gradient. The electron then passes through Cytcssa and back to the original chlorophyll. Alternatively, two excited electrons from chlorophyll P840* can go to two molecules of an iron- sulfur protein called ferredoxin. The two reduced ferredoxin molecules then reduce NAD" to NADH, using the catalytic power of an enzyme called ferredoxin-NAD reductase. In this case the electrons do not go back to the original chlorophyll. The two electrons are supplied by H28, which is oxidized to elemental sulfur. The two hydrogen ions from H28 are pumped across the cell membrane, contributing to the pH gradient that causes ATP synthesis. Proton gradient Green sulfur bacteria (Fe-S type) (b) ’ {b} Green sulfur bac- teria have two routes for electrons driven by excitation of P840: a cyclic route passes through a quinone to the cytochrome be? complex and back to the reaction center via cytochrome c, and a noocydic route from the reaction center through the iron-sulfur protein ferredoxin (Fd); then to NAB" in a reaction catalyzed by ferredoxinzNAD reductase. W31 5 Chloroplast structure Cyanobacteria and plant chloroplasts have combined, reorganized and added elements to photosystems l and H. (Cyan is a hue between blue and green). Thus it is appropriate to outline the structure of chloroplasts as it related to ATP synthesis. Chloroplasts are organelles. They have their own circular DNA Whose genes are similar to those of cyanobacteria. Below I have drawn an oversimplified cartoon of part of the chloroplast and then copied a more sophisticated diagram. The chloroplast has an outer and an inner membrane. Inside the inner membrane there is "stroma," which contains enzymes for the carbon fixing reactions that will be described in the next lecture. The light reactions take place in the "thylakoid membrane," which is a closed compartment with an inside called the "lumen" or “thylakoid space.” Thylakos means a sack or pouch in Greek. Although the thylakoid membrane consists of fancy interconnected stacks of compartments, my hand drawing represents it as a simple compartment. The rounded F1 portion of ATP synthase points out from the lumen into the stroma, which is arguably opposite of its arrangement in mitochondria. HTPQSQ outer harm lsl'ahe ‘l’l’lrlQKold ~ emigrant: inner Membrane. stroma lumen or JrkyloKo'xd space Inner membrane Outer membrane Thylakoid ‘- membrane lntezmembrane aroma ’ Thylakoid P 6 59399 iamellee Spam Discoveggf two reaction center electron transport Plants and cyanobacteria have two phosynthetic reaction centers. This fact was discovered using Chlorella, a green alga. The name means small green in Latin. The classical experiment investigates the wavelengths of light that cause algae to release oxygen. Starting at 660 nm, the quantum yield is about the same up to 690 nm, and then it drops to zero at 700 nm. However, in the presence of light in the range of 680 nm (yellow green light), light of 700 um. causes extra evolution of oxygen. This result suggested that there is a pigment that absorbs at 700 nm and one that absorbs at 680 nm? and that they can work together. 0.14 0.12 Presence (‘1 of yellow— o.1o green light 2 E .92 >‘ c... E 0.08 J E g c; 0.06 e O 0.04 Absence of yello‘ - green light 0.02 l L 660 680 700 720 Wavelength (nm) FIGURE 2444 Quantum yield for 02 production by Chlorella algae as a function of the wavelength of the incident light. The experiment was conducted in the absence (lower curve) and the presence (upper curve) of supplementary yellow-green light. The upper curve has been corrected for the amount of 02 production stimulated by the supplementary light alone. Note that the lower curve falls off precipitously above 680 run (the red drop). However, the supplementary light greatly increases the quantum yield in the wavelength range above 680 nm (far-red) in which the algae absorb light. {After Emerson, R., Chalmers, R.. and Cederstrand, C., Proc. Natl. Acad. Sci. 49, 137 (195?).] Pa? 7 FIGURE l9~56 integration of photosystems land I i in chloroplasts. This “‘2 scheme” shows the pathway of electron transicr from H20 dower £631} to NADF‘I {far right? in noncydlc photosynthesis The position on the verticai scale of each eiectron carrier reflects its standard reduction p0» teniéal, To raise the energy of electrons derived from i I30 to the energy levci required to reduce NADP‘L to NA "fitted" t phozoo z Rearrangement and reorganization of the photosystems The protein complexes that comprise these two photosystems, as well as the components that interconnect them, have been identified. Electrons enter the system from water at photosystem II, unlike the sulfur bacterial system that uses H2S as a source of electrons. Photosystem II splits water to produce oxygen, four electrons, and four hydrogen ions, which are pumped into the lumen, contributing to a pH gradient that causes ATP synthesis. Photosystem I accepts electrons from photosystem II and causes NADP+ to be reduced to NADPH, which provides biosynthetic reducing power. The cytochrome bsf complex, which is similar to complex III of mitochondria, connects the two photosystems. The cytochrome bsf complex pumps 2 H+ ions per electron into the thylakoid lumen, creating a pH gradient that causes ATP synthase to make ATP. The f stands for frons, the Latin word for leaf. Photosystem I @39 Photosystem II ~11} — Fag? Cyclic. Fd ._ ‘ ‘3’ \\ Fdd‘JADP+ '3; 7 QB oxidoreductase E _ Noncyclic F — Light Mi)?" [ [fl 0 _ ‘ NADPH / , Plastocyanin ‘ i ‘ fin?! Proton ' ' 9 gradient w 02. \ IPQA plastoquinone Mi lemivmg‘ 'PQB 2 second quinone \ bompfiggi rm “,7, 1‘0 --2102“hit'gi-ipiaqJ A0 : electron acceptor chlorool‘iyll A] : phylloquinone wwemw—wmw WWWWWMWNJ DWI, each electron must he I t “:eevy arrows; by photons absorbed in PSI? and P52, One ( , a A; 7 . t a ,. c i a r 3 per ezenirfifi in each gfshatrist‘aten} Airy? extrusion. dimes more are and ices NAUPH than the :toocvchc. page the higirehergy eiectrons flow “riowohih” through the carrier Chains shcmn. Protons move across the thylakoid membrane during the water— spiitring reaction and during electron transfer through the cytochrome bai'complex, producing the proton gradient that is essential to ATP for- mation An alternative path of electrons is cyclic electron transfer, in which electrons move from ferredoxm back lo the cytochrome inf com— giiexfi instead or reducing MAD?“ 2o NAFJPH‘ The cyciic pathway our HOW two H20 are transformed to 02 four electrons and two Hf Photosytem II in chloroplasts must transform two water molecules to a molecule of oxygen, four electrons and four hydrogen ions. One might imagine that this process would require four photons. When chloroplasts are left in the dark and then given very short pulses of light, no oxygen is evolved until the third pulse, which gives a large release of oxygen. Large releases are also observed for the seventh and eleventh pulses, so there is a four-pulse periodicity to oxygen release. FIGURE 24-21 The 02 yield per flash in darkuadapted spinach chloroplasts. Note that the yield peaks on the third flash and then on every fourth flash thereafter until the curve eventually damps out to its average value. [After Forbush, B., Kok, B.. and McGloin, M.P., Photochem. Photobiol. 14, 309 (1971).] 02 yield per flash 4 8 12 16 20 24 Flash number The special pair of chlorophyll molecules in photosystem II absorbs light best at 680 nm, so they are called P680. The excitation of an electron in P680, and its loss to a pheophytin is associated with one step of the oxidation of two water molecules. This occurs in an oxygen-evolving protein cemplex that contains four manganese ions, each of Which can have a valence from 2“” to 7“. The loss of four electrons to P680 from two waters yields oxygen plus four hydrogen ions, which are pumped into the thylakoid lumen, or inner thylakoid space. Electron paramagnetic resonance studies indicate that a tyrosine residue passes an electron to P680 and a hydrogen ion to the lumen, becoming a tyrosine radical. / 1 1st Exciton 2nd Exciton 8rd Egiciton 4th Exciton 3: Tyer ante—Lei. H V , L Lumen HGURE 39-62 Water-splitting activity of the exygenievclving complex. each abserptien causing the less of one electron from the Mn center, ere Shown here is the process that predaces a feur—electren exidizing dunes an oxidizing agent that can remove leer electrons from two moi . agent—~21 muiti nuclear center with several Mn i0ns~~in the wafer—splitting (tales of water. pe'educing Oz. The electrons lost from the Mn center pass (:empiex of PS“. The sequential abserptéon of few photons iexcitens}. one at a time is an oxidized Tyr residue in a P8” protein, than is Peae". "b g Q53 i’b «£3 The electron from PBSO", the excited form of P680, is transferred to pheophytin and then to a first plastoquinone (PQA), which is protein-bound and then to a second plastoquinone (PQB), in the same way that electron transfer occurs in purple bacteria. These quinines are called plastoquinones, because they are in chloroplasts. PQB eventually accepts a second electron and then takes up two hydrogen ions, in this case from the stroma. These PQBHZ molecules exchange with membrane-bound quinones. Electrons from the quinone pool are transferred to a cytochrome 63f complex. The bef complex is similar to cytochrome 1901 in purple bacteria and to Complex III of mitochondria. When an electron passes through the (36f complex, two hydrogen ions are pumped from the stroma to the lumen. When four electrons from two water molecules are passed through this complex, about 8 hydrogen ions are pumped from the stroma to the thylakoid lumen. Each electron is then passed to plastocyanin, which is a copper-protein found at the lumenal surface of the membrane. The bound copper ion can have the +2 or +1 valence. The proteins in photosystem II have similar amino acid sequences to those found in purple bacteria. Plastocyanin passes its electron to the oxidized form of P700 in Photosystem I, which contains a special pair of chlorophylls that absorb maximally at 700 nm. When P700 is activated by a photon, it loses an electron to a chain of carriers, and it regains the electron from plastocyanin. The carriers in the chain are another chlorophyll (A0), phylloquinone (A1 in the diagram, also called Vitamin K1. Phyllon means “leaf ’), and then to an iron-sulfur cluster. Eventually, the electron gets to an iron-protein called ferredoxin. Two ferredoxins give two electrons to an enzyme called ferredoxin-NADP+ reductase. The FAD prosthetic group of this protein becomes reduced to FADH2, which then reduces NADP+ to NADPH + H". The overall reaction is thus 01 ZHZO + 2 NADP+ —) 02 + 2 NADPH + 2H+ (i /CH3 Cl E~c—N\ This equation ignores the pH gradient and ATP synthesis. CH3 3-(3,4—Dichlorophenyl)-1,1-dimethylurea The target of a herbicide (DCMU) The herbicide 3-(3,4-dichlorophenyl)-1,1—dimethylurea (DCMU) prevents the oxidized cytochrome bsf complex from accepting electrons from plastoquinone QB. Cyclic photophosphoryjlation When NADPH predominates and NADP+ is almost depleted, electrons can flow from photosystem I through ferredoxin to the cytochrome be)” complex, causing hydrogen ions to be pumped into the thylacoid lumen. This causes ATP to be synthesized. The passing of electrons from PSI back to cytochrome bef is called cyclic photophosphorylation. Daniel Arnon at Berkeley Page 10 discovered cyclic photophorphorylation in broken chloroplasts that could synthesize ATP Without liberation of oxygen. ATP synthesis, and guantitation of the light reactions During the cleavage of two waters to ox...
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