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Biochemistry A H. Y. Sung III. Oxidative Phosphorylation and Photophosphorylation 1. INTRODUCTION A. Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organisms. Oxidative degradation of carbohydrates, fats and amino acids drives the synthesis of ATP. B. Photophosphorylation : photosynthetic organisms capture the energy of light (the ultimate source of energy in the biosphere) and make ATP. C. Oxidative phosphorylation and photophosphorylation account for most of the ATP synthesized by most organisms most of the time. D. In eukaryotes, oxidative phosphorylation occurs in mitochondria. Photophosphorylation in chloroplasts. E. Oxidative phosphorylation involves the reduction of O2 to H2O with electrons donated by NADH and FADH2 , and occurs equally well in light or darkness. F. Photophosphorylation involves the oxidation of H2O to O2 with NADP+ as ultimate electron acceptor, it is absolutely dependent on the energy of light. G. These two highly efficient energy-converting processes have fundamentally similar mechanisms. The energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton motive force. H. Chemiosmotic theory for ATP synthesis : P. Mitchell in 1961. 2. OXIDATIVE PHOSPHORYLATION A. The mitochondria A.1 The site of eukaryotic oxidative metabolism a. Pyruvate dehydrogenase, citric acid cycle enzymes. b. Enzymes catalyzing urea cycle and fatty acid oxidation. c. Enzymes and redox proteins involved in electron transport and oxidative phosphorylation. d. As the cells’ “ power plant ”. A.2 Mitochondrial anatomy a. Mitos : thread ; chondros ; granule. b. Mitochondria vary in size and shape, they are often ellipsoidal with dimensions of around 0.5 × 1.0 µm. c. A eukaryotic cell contains approximately 2000 mitochondria, occupy roughly 1/5 of its total cell volume. d. The structure of mitochondria. Page 1 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung e. The components of the matrix. A.3 Mitochondrial transport systems a. Outer membrane contains porins : proteins that permit the free diffusion of molecules of up to 10 kD. b. Intermembrane space. c. Inner membrane : • ~ 75% protein by mass. ‚ Twice the density of embedded particles as the outer membrane. ƒ Freely permeable only to O2 , CO2 and H2O. „ Respiratory chain proteins and numerous transport proteins. … The controlled impermeability. B. Electron transfer reactions in mitochondria B.1 Electrons are funneled to universal electron acceptors. a. Oxidative phosphorylation begins with the entry of electrons into the respiratory chain. b. Nicotinamide nucleotide-linked dehydrogenases : NAD+ or NADP+ as electron acceptors. • Most dehydrogenases that act in catabolism are specific for NAD+ in cytosol or mitochondria, some have mitochondrial and cytosolic isozymes (malate dehydrogenase). ‚ Nicotinamide nucleotide transhydrogenase ƒ The biological functions of NADH and NADPH. NADH : catabolic reaction NADPH : anabolic reaction c. Flavoproteins contain a very tightly, sometimes covalently, bound flavin nucleotide, either FMN or FAD. • The oxidized flavin nucleotide can accept either one electron (semiquinone form) or two (FADH2 or FMNH2). ‚ The standard reduction potential of a flavin nucleotide depends on the protein with which it is associated. B.2 Electrons pass through a series of membrane-bound carriers. a. Three types of electron transfer occur in oxidative phosphorylation. • direct transfer of electrons as in the reduction of Fe 3+ to Fe2+. - ‚ transfer as a hydrogen atom (H + + e ). - ƒ transfer as a hydride ion (:H , two electrons). b. Five types of electron-carrying molecules function in the respiratory chain. • Nicotinamide nucleotide ‚ Flavoproteins ƒ Ubiquinone (hydrophobic quinone) Page 2 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung „ Cytochromes (iron-containing proteins) … Iron-sulfur proteins c. Ubiquinone (coenzyme Q) • A lipid-soluble benzoquinone with a long isoprenoid side chain (Q 10, Q6, Q8) : ubiquity in respiring organisms. ‚The closely related compounds : plastoquinone (plant chloroplasts) and menaquinone (bacteria). ƒ Ubiquinone can accept one electron to become the semiquinone radical (QH+) or two electrons to form ubiquinol. „ Ubiquinone is small and hydrophobic, it is freely diffusible within the lipid bilayer of inner mitochondrial membrane. d. Cytochromes • Redox-active proteins occur in all organisms except a few types of obligate anaerobes. ‚ Proteins contain heme groups and iron. The heme groups of the reduced Fe (II) cytochromes have prominent visible absorption spectra consisting of 3 peaks : α, β and γ (Soret) bands. ƒ The wavelength of the α-peak is used to differentiate the various cytochromes. „ Heme groups. (a) b-type cytochromes : protoporphyrin IX. (b) c-type cytochromes : vinyl groups linked with Cys sulfhydryls to form thioether linkages. (c) a-type cytochromes : a long hydrophobic tail of isoprene units ; formyl group in place of methyl substituent in hemes b and c. … The heme cofactors of a and b cytochromes are tightly, but not covalently, bound to their associated proteins, the hemes of c-type cytochromes are covalently attached through Lys residues. † The standard reduction potential of the heme iron atom of a cytochrome depends on its interaction with protein side chains and is different for each cytochrome. e. Iron-sulfur proteins (non-heme iron proteins) • Iron is present not in heme but in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both. ‚ Four types of iron-sulfur proteins. (a) Single Fe ion (b) 2 Fe-2S (c) 4 Fe-4S (d) Rieske iron-sulfur proteins ƒ All iron-sulfur proteins participate in one-electron transfers in which one iron atom is oxidized or reduced. Page 3 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung f. Methods for determining the sequence of electron carriers. • The activity of the electron-transport chain : O2 consumption. ‚ The sequence of electron carriers roughly reflects their relative reduction potentials. ƒ Difference spectra. „ Specific inhibitors. (a) Rotenone and amytal (Complex I) (b) Antimycin A and myxothiazol (Complex III) (c) Cyanide, azide and carbon monooxide (Complex IV) B.3 Electron carriers function in multienzyme complexes a. The electron carriers of the respiratory chain are organized into membrane-embedded supramolecular complexes. b. Separation of functional complexes of the respiratory chain • The outer mitochondrial membrane is first removed by treatment with the detergent digitonin. ‚ Fragments of inner membrane : osmotic rupture and gently dissolved in a second detergent. ƒ Four different complexes and ATP synthase (Complex V, in vitro, only ATPhydrolyzing, not ATP-synthesizing activity) are obtained by ion-exchange chromatography. c. Protein components of the mitochondrial electron-transfer chain d. Path of electrons from NADH, succinate, fatty acyl-CoA and glycerol 3-phosphate to ubiquinone. e. Complex I (NADH : ubiquinone oxidoreductase) : NADH to ubiquinone • The largest protein complex in the inner membrane (a) 42 polypeptides with a total mass of 850 kD (b) 1 molecule FMN and 6 to 7 iron-sulfur clusters ‚ The coenzymes of Complex I (a) Iron-sulfur proteins (nonheme iron proteins) : [2Fe-2S] and [4Fe-4S] clusters ; one-electron oxidation and reduction (b) FMN (3 states) : tightly bound to protein (c) CoQ (3 states) : hydrophobic tail (Q10 , Q6 , Q8) ƒ Electron transfer in Complex I „ Proton translocation : four protons are translocated from the matrix to the intermembrane space, as electrons are transferred between the redox centers of Complex I. f. Complex II (succinate-CoQ oxidoreductase) : succinate to ubiquinone • Succinate dehydrogenase and 3 small hydrophobic subunits. 5 subunits and 127 kD. ‚ Redox groups : FAD, one [4Fe-4S] cluster, two [2Fe-2S] clusters, cytochrome b560. ƒ Acyl-CoA dehydrogenase-ETF-ubiquinone oxidoreductase and glycerol 3-phosphate Page 4 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung dehydrogenase. „ Complexes I and II do not operate in series, both accomplish the same result : transfer of electrons to CoQ from reduced substrates (NADH or succinate). … CoQ serves as a sort of collection point for electrons. g. Complex III (cytochrome bc1 complex or ubiquinone : cytochrome c oxidoreductase) : ubiquinone to cytochrome c • 2 b-type cytochromes (b562, b566), cytochrome c1, [2Fe-2S] cluster (Rieske center). ‚ A dimer with 11 subunits in each 248 kD monomer (bovine heart). ƒ Electron transport in Complex III : The Q cycle. (a) One molecule CoQH2 (two-electron carrier) to reduce 2 molecules of cytochrome c (one-electron carrier). (b) CoQH2 undergoes a two-cycle reoxidation in which the semiquinone is a stable intermediate. (c) The first and second cycle reactions. (d) The overall reaction : CoQH2 is oxidized, two reduced cytochrome c molecules and four protons appear on the outer site of the membrane. (e) Inhibitors : myxothiazol and antimycin A. „ Cytochrome c : a soluble electron carrier. h. Complex IV (cytochrome oxidase) : cytochrome c to O2 • The reaction ‚ A dimer with 13 subunits in each 204 kD monomer (mammalian cell). ƒ Subunits I, II and III are encoded by mitochondrial DNA, the remaining subunits are nuclearly encoded and transported into the mitochondria. „ Four redox centers : cytochrome a, a3, CuB and CuA center. … Reduction of O2 by cytochrome c oxidase. (a) The reduction of O2 to 2 H2O takes place at the cytochrome a3-CuB binuclear complex. (b) The cytochrome c oxidase reaction. (c) Four protons are consumed in the reduction of O2 by cytochrome c oxidase. These protons originate in the mitochondrial matrix. i. Summary of the flow of electrons and protons through the four complexes of the respiratory chain. B.4 Some agents that interfere with oxidative phosphorylation or photophosphorylation B.5 The energy of electron transfer is efficiently conserved in a proton gradient a. NADH oxidation is highly exergonic : oxidation of 1 mole of NADH by O2 releases 220 kJ of free energy (succinate : 150 kJ/mole). b. The flow of electrons results in the pumping of protons across the mitochondrial inner membrane, making the matrix alkaline relative to the intermembrane space. c. This proton gradient provides the energy (proton-motive force) for ATP synthesis from Page 5 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung ADP + Pi. B.6 Plant mitochondria have alternative mechanisms for oxidizing NADH a. Plant mitochondria supply ATP during periods of low illumination or darknees by mechanisms entirely analogous to those used by nonphotosynthetic organisms. b. In the light, the principal source of mitochondrial NADH is a reaction in which glycine (photorespiration) is converted to serine. c. NADH → ubiquinone → O2 d. Cyanide-resistant NADH oxidation (QH2 oxidase is not inhibited by cyanide) B.7 Alternative respiratory pathways and hot, stinking plants a. Heat production (thermogenesis) in some flowering plants. b. Normal electron-transfer system c. Cyanide-resistant QH2 oxidase transfers electrons from the ubiquinone pool directly to oxygen. Energy is released as heat. d. Two alternative NADH dehydrogenases • directly transfers electrons from NADH to ubiquinone, insensitive to the Complex I inhibitor rotenone. ‚ external NAD(P)H dehydrogenase (on the external face of the inner membrane). C. ATP synthesis C.1 The endergonic synthesis of ATP from ADP and Pi in mitochondria is catalyzed by an ATP synthase (Complex V) that is driven by the electron-transport process. C.2 The free energy released by electron transport through Complexes I-IV must be conserved in a form that ATP synthase can use. C.3 The chemiosmotic model a. Mitchell’s theory. • Oxidative phosphorylation requires an intact inner mitochondrial membrane. ‚ The inner membrane is impermeable to ions such as H + , OH- , K+ and Cl- ƒ Creation of electrochemical gradient „ Uncoupler : allow electron transport but inhibit ATP synthesis b. Electron transport generates a proton gradient • Elctron transport causes Complex I, III, IV to transport protons across the inner + mitochondrial membrane from the matrix, a region of low [H ] to the intermembrane space, a region of high [H + ]. ‚ Proton motive force powers ATP synthesis. ƒ Three protons are required per ATP synthesis. c. Coupling of electron transfer and ATP synthesis in mitochondria d. Evidence for the role of a proton gradient in ATP synthesis e. Uncoupling oxidative phosphorylation • Electron transport and oxidative phosphorylation are tightly coupled. Page 6 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung ‚ Uncoupler (uncouple electron transport and ATP synthesis) : 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Both also uncouple photophosphorylation. ƒ Action of uncoupler : proton-transporting ionophore. „ Allow electron transport but inhibit ATP synthesis. C.4 ATP synthase has two functional domains F 0 and F1. a. Mitochondrial ATP synthase is an F-type ATPase similar in structure and mechanism to the ATP synthases of chloroplasts and eubacteria. b. Multisubunit transmembrane protein (450 kD) : inner mitochondrial membrane. c. Two functional units : F0 and F1. • F0 (oligomycin-sensitive) : water-insoluble transmembrane proton channel (at least 8 different types of subunits). ‚ F1 (first factor as essential) : water-soluble peripheral membrane protein (5 types of subunits). Isolated F1 hydrolyzes ATP but can not synthesize ATP. C.5 ATP is stabilized relative to ADP on the surface of F1 From the binding constants show that F0F1 binds ATP with very high affinity and ADP with much lower affinity. C.6 The proton gradient drives the release of ATP from the enzyme surface. a. Reaction coordinate diagrams for ATP synthase and a typical enzyme. b. ATP synthase must cycle between a form that binds ATP very tightly and a form that releases ATP. C.7 Each β subunit of ATP synthase can assume three different conformations. a. Mitochondrial F1 has nine subunits of five different types, with the composition α3β3γδε. b. Each of the three β subunits has one catalytic site for ATP synthesis. c. F0 complex is composed of three subunits a, b and c, in the proportion ab2c10-12. Subunit c is a small (8 kD) very hydrophobic polypeptide. d. The structure of the F0F1 complex. C.8 Rotational catalysis is key to the binding-change mechanism for ATP synthesis. a. Binding-change model for ATP synthase. b. Rotation of F0 and γ experimentally demonstrated. C.9 Chemiosmotic coupling allows nonintegral stoichiometries of O2 consumption and ATP synthesis. a. P/O ratio, P/2e ratio. b. Measurement of P/O ratio in intact mitochondria. c. P/O ratio is 3 for NADH and 2 for FADH 2. d. P/O ratio is 2.5 for NADH and 1.5 for FADH 2 (succinate). C.10 The proton-motive force energizes active transport. Page 7 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung a. The primary role of the proton gradient in mitochondria is to furnish energy for synthesis of ATP, the proton-motive force also drives several transport processes essential to oxidative phosphorylation. b. The inner mitochondrial membrane is generally impermeable to charged species. 3- c. Adenine nucleotide translocase (antiporter) : ADP outward. It is specifically inhibited by atractyloside. 4- into the matrix and ATP - d. Phosphate translocase (symporter) : one H 2PO4 and H+ into the matrix. C.11 Shuttle systems are required for mitochondrial oxidation of cytosolic NADH. a. NADH dehydrogenase of the inner mitochondrial membrane of animal cells accepts electrons only from NADH in the matrix. b. Malate-aspartate shuttle in liver, kidney and heart. Malate-αKG transporter and glutamate-aspartate transporter. c. Glycerol 3-phosphate shuttle in skeletal muscle and brain. d. Externally oriented NADH dehydrogenase transfers electrons directly from cytosolic NADH into the respiratory chain at the level of ubiquinone in the mitochondria of higher plants. D. Regulation of oxidative phosphorylation D.1 Introduction a. Oxidative phosphorylation produces most of the ATP made in aerobic cells. b. Glycolysis under anaerboic conditions yields only 2 ATP per glucose. c. Complete oxidation of a molecule compound to CO 2 • Glucose : 30 or 32 ATP ‚ Palmitoyl-CoA : 108 ATP D.2 Oxidative phosphorylation is regulated by cellular energy needs a. The rate of respiration in mitochondria is limited by the availability of ADP as a substrate for phosphorylation. b. Acceptor control and acceptor control ratio. c. Mass-action ratio of the ATP-ADP system : [ATP]/[ADP][Pi] D.3 Uncoupled mitochondria in brown fat produce heat a. Heat generation is the physiological function of brown adipose tissue (brown fat), which contains numerous mitochondria whose cytochromes cause its brown color. b. Nonshivering thermogenesis. c. The mechanism of heat generation in brown fat involves the regulated uncoupling of oxidative phosphorylation. d. Uncoupling protein (UCP ; thermogenin) : in inner mitochondrial membrane. e. The flow of protons through UCP is inhibited by physiological concentrations of purine nucleotides (ADP, ATP, GDP, GTP), but this inhibition can be overcome by free fatty acids. Page 8 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung D.4 ATP-producing pathways are coordinately regulated. a. The major catabolic pathways have interlocking and concerted regulatory mechanisms that allow them to function together in an economical and self-regulating manner to produce ATP and biosynthetic precursors. b. The relative concentrations of ATP and ADP control the rates of electron transfer, oxidative phosphorylation, citric acid cycle, pyruvate oxidation and glycolysis. c. ATP is an allosteric inhibitor of phosphofructokinase-1 and pyruvate dehydrogenase. d. Phosphofructokinase-1 is inhibited by ATP and citrate. D.5 Mutations in mitochondrial genes cause human disease. a. Mitochondria contain their own genome, a circular double-stranded DNA molecule. b. Human mitochondrial chromosome contains 37 genes (16,569 bp), including 13 that encode subunits of proteins of the respiratory chain, the remaining genes code for rRNA and tRNA molecules. c. Many mitochondrial proteins are encoded by nuclear genes. d. Leber’s hereditary optic neuropathy : Arg → His e. Myoclonic epilepsy and ragged-red fiber disease : leucyl-tRNA. D.6 Mitochondria probably evolved from endosymbiotic bacteria a. The existence of mitochondrial DNA, ribosomes and tRNAs supports the theory of the endosymbiotic origin of mitochondria. b. Bacterial respiratory chain c. Rotation of bacterial flagella by proton-motive force 3. PHOTOSYNTHESIS : HARVESTING LIGHT ENERGY A. Introduction A.1 Solar energy is the ultimate source of all biological energy. A.2 Photosynthetic and heterotrophic organisms live in a balanced steady state in the biosphere. A.3 Photosynthesis occurs in a variety of bacteria and in unicellular eukaryotes (algae) as well as in higher plants. A.4 CO2 + H2O → O2 + (CH2O) H2O donates electrons (as hydrogen) for the reduction of CO 2 to carbohydrate. B. General features of photophosphorylation B.1 Photophorylation vs oxidative phosphorylation a. Photosynthesis, in which light energy drives the reduction of carbon, is essentially the reverse of oxidative carbohydrate metabolism. b. Photophosphorylation differs from oxidative phosphorylation in requiring the input of energy in the form of light to cerate a good electron donor. Page 9 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung c. In photophosphorylation, electrons flow through a series of membrane-bound carries including cytochromes, quinones and iron-sulfur proteins, which protons are pumped across a membrane to create an electrochemical potential. d. ATP synthesis is catalyzed by a membrane-bound ATP synthase complex closely similar to that of oxidative phosphorylation. B.2 Two stages of photosynthesis a. Light reactions (light-dependent) : specialized pigment molecules capture light energy and are oxidized. Electrons are transferred to generate NADPH and a transmembrane proton gradient that drives ATP synthesis. The oxidized pigment molecules are reduced by H2O and generating O2. b. Carbon-assimilation reactions (carbon-fixation reactions, dark reactions) : NADPH and ATP produced by the light reactions are used to incorporate CO2 into carbohydrates and other products. B.3 Photosynthesis in higher plants takes place in chloroplasts. a. The site of photosynthesis in eukaryotes (algae and higher plants). Cells contain 1 to 1000 chloroplasts, vary in size and shape, ~ 5-µm-long ellipsoids. b. Chloroplast anatomy • Highly permeable outer membrane and nearly impermeable inner membrane and intermembrane space. ‚ Stroma : The concentrated solution of enzymes, small molecules and ions in the interior of a chloroplast ; the site of carbohydrate synthesis. The stroma also contains DNA, RNA and ribosomes involved in the synthesis of several chloroplast proteins. ƒ Thylakoid : The innermost compartment in chloroplasts, which is formed by invagination of innermembrane. The thylakoid membrane is the site of light reactions. „ Grana : The stacked disks of the thylakoid in a chloroplast; 10-100 grana/chloroplast. B.4 Light drives electron flow in chloroplasts a. Hill reaction light 2 H20 + 2 A → 2 AH2 + O2 b. Hill reagent : 2,6-dichlorophenolindophenol c. CO2 is not required and O2 evolution is dissociated from CO2 reduction. + d. NADP is the biological electron acceptor in chloroplasts. C. Light absorption C.1 The interaction of light and matter a. Planck’s law E = hv = hc/λ b. Visible light is electromagnetic radiation of wavelengths 400 to 700 nm. c. The spectrum of electromagnetic radiation and the energy of photons in the visible Page 10 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung range of the spectrum. C.2 Chlorophylls absorb light energy for photosynthesis a. Principal photoreceptor is chlorophylls : green pigments with polycyclic planar tetrapyrrole. 2+ b. Chlorophyll structures : Mg • Chlorophyll a and b are green; absorption spectra are different : in plants. ‚ Phytol side chain ƒ Bacteriochlorophyll a and b: in algae and photosynthetic bacteria. c. Light-harvesting complexes (chlorophyll is associated with specific binding proteins ) contain multiple pigments. • Primary reactions take place at photosynthetic reaction centers. ‚ Chlorophyll molecules do not participate directly in photochemical reaction, but function to gather light and they act as light-harvesting antennas. ƒ Phycobilins : phycoerythrobilin (red) and phycocyanobilin (blue) : open-chain tetrapyrroles, cyanobacteria and red algae (water-dwelling photosynthetic organisms). „ Phycobilisomes : phycobiliproteins C.3 Accessory pigments (secondary light-absorbing pigments) extend the range of light absorption. a. Carotenoids : components of all green plants and many photosynthetic bacteria. • β-Carotene : red-orange isoprenoid ‚ Lutein (xanthophyll) : yellow b. Action spectrum C.4 Chlorophyll funnels absorbed energy to reaction centers by exciton transfer. a. Photosystems : in spinach chloroplasts, each photosystem contains about 200 molecules of chlorophylls and 50 molecules of carotenoids. • Photochemical reaction center ‚ Light-harvesting or antenna molecules b. Organization of photosystems in the thylakoid membrane c. Exciton transfer D. The central photochemical event : light-driven electron flow D.1 Light-driven electron transfer in plant chloroplasts occur in the thylakoid membrane. D.2 Bacteria have one of two types of single photochemical reaction centers a. Bleaching of the pigments : P870, P680 and P700 b. Electrons pass through pheophytin to a quinone : in purple bacteria. c. Electrons pass through quinone to an iron-sulfur center : in green-sulfur bacteria. D.3 The pheophytin-quinone reaction center (type II reaction center) 2+ a. Pheophytin : chlorophyll lacking the central Mg , replaced by two protons. Page 11 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung b. Three basic modules. • Single reaction center (P870) ‚ Cytochrome bc1 electron-transfer complex ƒ ATP synthase (proton gradient) c. Photoreaction center of the purple bacterium D.4 The Fe-S reaction center (Type- I reaction center) a. P840 and H2S b. Cyclic route : electrons → quinone → cytochrome bc1 complex → cytochrome c c. Noncyclic route : electrons → ferredoxin → NAD+ (ferredoxin- NAD+ reductase) → NADH D.5 In higher plants, two reaction centers act in tandem a. Two systems have distinct and complementary functions. a.1 Photosystem I (PSI, ferredoxin type) : • reduces NADP+ to NADPH ‚ red light (P700) ƒ high ratio of chlorophyll a to chlorophyll b a.2 Photosystem II (PSII, pheophytin-quinone type) • oxidizes H2O to release oxygen ‚ yellow-green light (P680) ƒ roughly equal amounts of chlorophyll a and b a.3 Oxygenic and anoxygenic photosynthesis a.4 All O2-evolving photosynthetic cells (higher plants, algae and cyanobacteria) contain both PSI and PSII. b. Z scheme : 2 H2O + 2 NADP+ + 8 photons → O2 + 2 NADPH + 2 H+ • Integration of PSI and PSII in chloroplasts The overall reaction initiated by light in PSII : 4 P680 + 4 H+ + 2 PQB + 4 photons → 4 P680+ + 2 PQBH2 ‚ Commerical herbicides block electron transfer through the cytochrome b6f complex and prevent ATP production. ƒ PSI Ferredoxin (10.7 kD ; 2Fe-2S center) and ferredoxin : NADP+ oxidoreductase D.6 Spatial separation of PSI and PSII prevents exciton larceny a. Localization of PSI and PSII in thylakoid membranes b. Modulation of granal stacking equalizes electron flow in PSI and PSII. D.7 The cytochrome b6f complex links PSI and PSII a. Cytochrome b6f complex • b-type cytochrome with two heme groups (bH and bL) ‚ Rieske iron-sulfur protein (20 kD) Page 12 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung ƒ c-type cytochrome c552 (cytochrome f) b. Electron and proton flow through the cytochrome b6f complex D.8 Cyanobacteria use the cytochrome b6f complex and cytochrome c in both oxidative phosphorylation and photophosphorylation. a. Cyanobacteria do not have mitochondria or chloroplasts. b. Dual roles of cytochrome b6f and cytochrome c in cyanobacteria D.9 Water is split by the oxygen-evolving complex. a. The source of the electrons passed to NADPH in plants is water. b. A variety of electron donors for photosynthetic bacteria : acetate, succinate, malate or sulfide. c. Two water molecules are split and four photons are required. - 2 H 2O → 4 H + + 4 e + O 2 d. Water-splitting activity of the oxygen-evolving complex (water-splitting complex) E. ATP synthesis by photophosphorylation E.1 Light reaction a. NADPH and ATP are produced. b. Photophosphorylation : light-dependent ATP production in pigment-containing membranes structures (chromatophores). E.2 A proton gradient couples electron flow and phosphorylation a. The similarity between oxidative phosphorylation and photophosphorylation • The reaction centers, electron carriers and ATP-forming enzymes are located in a proton-impermeant membrane (thylakoid membrane). ‚ Uncoupler effects photophosphorylation ƒ Photophosphorylation can be blocked by venturicidin and similar agents that inhibit mitochondrial ATP synthase. „ F0F1 complexes of the thylakoid membrane are very similar to that of mitochondria. b. Proton and electron circuits in thylakoids c. Jagendorf’s experiment E.3 The approximate stoichiometry of noncyclic photophosphorylation 2 H2O + 8 photons + 2 NADP+ + ~ 3 ADP + ~ 3 Pi → O2 + ~ 3 ATP + 2 NADPH E.4 Cyclic electron flow produces ATP but not NADPH or O 2 a. Cyclic electron flow involves only PSI. b. Electrons passed from P700 move back through the cytochrome b6f complex to plastocyanin. light c. Cyclic photophosphorylation : ADP + Pi → ATP + H2O d. Cyclic pathway : cyclic electron flow increases the level of ATP synthesis relative to that of NADPH. Page 13 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com Biochemistry A H. Y. Sung E.5 The ATP synthase of chloroplasts is like that of mitochondria. a. Two functional components : CF0 and CF1 b. CF0 : a transmembrane proton pore c. CF1: a peripheral membrane protein complex d. The mechanism of chloroplast ATP synthase is identical to that of mitochondrial analog. E.6 Chloroplasts probably evolved from endosymbiotic bacteria a. Like mitochondria, chloroplasts contain their own DNA and protein-synthesizing machinery. b. Some of the polypeptides of chloroplast proteins are encoded by chloroplast genes and synthesized in the chloroplast : others are encoded by nuclear genes. c. Prochlorophytes : photosynthetic bacteria contain chlorophyll a and b but not phycobilins, are the likely progenitors of the chloroplasts of modern higher plants. Evolutionary relationship. E.7 Diverse photosynthetic organisms use hydrogen donors other than water a. At least half of the photosynthetic activity on earth occurs in microorganisms algae, other photosynthetic eukaryotes and photosynthetic bacteria. b. Cyanobacteria have PSII and PSI in tandem. Other groups of photosynthetic bacteria have single reaction centers and do not split H2O or produce O2. c. Obligate anaerobes cannot tolerate oxygen and use some other compound other than H2O as electron donor. d. Green sulfur bacteria use hydrogen sulfide. e. Some photosynthetic bacteria use organic compounds such as lactate. f. General form of photosynthesis : light 2 H2D + CO2 → (CH2O) + H2O + 2D E.8 In halophilic bacteria, a single protein absorbs light and pumps protons to drive ATP synthesis. a. The halophilic bacterium Halobacterium salinarum traps the energy of sunlight in a process very different from the other photosynthetic organisms. b. The environment of bacterium living : brine ponds and salt lakes. c. The organisms are aerobes and normally use O2 to oxidize organic fuel molecules. d. Bacteriorhodopsin (26 kD ; 247 amino acid residues) : light-absorbing pigment contains retinal. e. Light-driven proton pumpimg by bacteriorhodopsin. f. ATP synthesis. g. The halobacteria do not evolve O2 and carry out photoreduction of NADP+. Page 14 PDF created with FinePrint pdfFactory Pro trial version http://www.fineprint.com