Atp synthesis
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  • 1. ATP SYNTHESIS Centre for Nano science and Technology Course: Biology for Nanotechnology. Code: NST 623 Course instructor: Dr. S.Kannan. PRESENTED BY ROOPAVATH UDAY KIRAN M.Tech 1st year
  • 2. Outline • Introduction • Electron-Transfer Reactions in Mitochondria • ATP Synthesis • Regulation of Oxidative Phosphorylation • General Features of Photophosphorylation • Light Absorption • The Central Photochemical Event: Light- Driven Electron Flow • ATP Synthesis by Photophosphorylation
  • 3. Adenosine Triphosphate Energy source photosynthesis and cellular respiration Signal transduction second messenger cAMP DNA replication AMP
  • 4. Structure Purine base 1’C 5’C Pentos sugar Three phosphate groups
  • 5. • Substrate-level phosphorylation direct transfer of a phospate group to ADP
  • 6. In mitochondrion
  • 7. • Chemiosmotic Phosphorylation Electrochemical gradient + Osmosis 1.Oxidative Phosphorylation 2. Photophosphorylation
  • 8. ATP is synthesized using the same strategy in oxidative phosphorylation and photophosphorylation • Oxidative phosphorylation is the process in which ATP is generated as a result of electron flow from NADH or FADH2 to O2 via a series of membrane-bound electron carriers, called the respiratory chain (reducing O2 to H2O at the end). • Photophosphorylation is the process in which ATP (and NADPH) is synthesized as a result of electron flow from H2O to NADP+ via a series of membrane-bound electron carriers (oxidizing H2O to O2 at the beginning).
  • 9. • Oxidative phosphorylation and photophosphorylation are mechanistically similar in three respects. (1) Both processes involve the flow of electrons through a chain of membrane-bound carriers. (2) The free energy made available by this ―downhill‖ (exergonic) electron flow is coupled to the ―uphill‖ transport of protons across a proton-impermeable membrane, conserving the free energy of fuel oxidation as a. transmembrane electrochemical potential (3) The transmembrane flow of protons down their concentration gradient through specific protein channels provides the free energy for synthesis of ATP, catalyzed by a membrane protein complex (ATP synthase) that couples proton flow to phosphorylation of ADP.
  • 10. ATP GenerationGlycolysis • Conversion of glucose to pyruvate • Net synthesis of 2 ATP by substrate level phosphorylation Krebs Cycle • Converts pyruvate to acetyl CoA & carbon dioxide • 10 molecules of coenzymes NADH and 2 of FADH2 are produced. Results in synthesis of 30 ATP and 4 ATP molecules, respectively in the respiratory chain. Electron Transport (Respiratory) Chain • The reduced coenzymes enter into the respiratory chain of the inner mitochondrial membrane • Electron transport along the chain generates a proton electrochemical gradient and this is used to produce ATP
  • 11. Chemiosmotic theory: • Introduced by Peter Mitchell in 1961 • Transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions. • It provides insight into the processes of oxidative phosphorylation and photophosphorylation, and into such apparently disparate energy transductions as active transport across membranes and the motion of bacterial flagella.
  • 12. Proton Gradient Across the Membrane: “Chemiosmosis” • It is the universal mechanism of ATP production which involves the production of a proton motive force (pmf) based on a proton gradient across the membrane. • Energy to establish this electrochemical proton gradient is provided by the energy released as electrons move to lower energy levels down the electron transport chain and the coupling of this free energy to the movement of protons across the IMM against the proton gradient [from matrix to IMS] • ATP is synthesized by the ATP synthase FoF1 complex : protons move with the proton gradient through FoF1 to generate ATP [from IMS to matrix]
  • 13. The chemiosmotic model of Mitchell
  • 14. OXIDATIVE PHOSPHORYLATION • The discovery in 1948 by Eugene Kennedy and Albert Lehninger that mitochondria are the site of oxidative phosphorylation in eukaryotes marked the beginning of the modern phase of studies in biological energy transductions. • Oxidative phosphorylation begins with the entry of electrons into the respiratory chain. • Most of these electrons arise from the action of dehydrogenases that collect electrons from catabolic pathways and funnel them into universal electron acceptors—nicotinamide nucleotides (NAD+ or NADP+) or flavin nucleotides (FMN or FAD).
  • 15. • The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. • Three types of electron transfers occur in oxidative phosphorylation: (1) Direct transfer of electrons, as in the reduction of Fe+3 to Fe+2; (2) Transfer as a hydrogen atom (H+ +e); and (3) Transfer as a hydride ion (:H), which bears two electrons. • The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation- reduction reaction.
  • 16. Electrons collected in NADH and FADH2 are released and transported to O2 via the respiratory chain • The chain is located on the convoluted inner membrane (cristae) of mitochondria in eukaryotic cells (revealed by Eugene Kennedy and Albert Lehninger in 1948) or on the plasma membrane in prokaryotic cells. • A 1.14-volt potential difference (E`0) between NADH (-0.320 V) and O2 (0.816 V) drives electron flow through the chain.
  • 17. • The respiratory chain consists of four large multi- protein complexes (I, II, III, and IV; three being proton pumps) and two mobile electron carriers, ubiquinone (Q or coenzyme Q, and cytochrome c. • Prosthetic groups acting in the proteins of respiratory chain include flavins (FMN, FAD), hemes (heme A, iron protoporphyrin IX, heme C), iron-sulfur clusters (2Fe-2S, 4Fe-4S), and copper.
  • 18. Four multi-protein Complexes (I, II, III, and IV) Two mobile Electron carriers I II III IV
  • 19. • Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble benzoquinone with a long isoprenoid side chain • Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement.
  • 20. Complete reduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate.
  • 21. Heme groups of cytochrome proteins Heme groups Of cytochromes
  • 22. Different types of iron-sulfur centers •Iron atoms cycle between Fe2+ (reduced) and Fe3+(oxidized). •At least eight Fe-S proteins act in the respiratory chain. 4Fe-4S2Fe-2S A ferredoxin
  • 23. NADH:Ubiquinone Oxidoreductase a.k.a. Complex I • One of the largest macro- molecular assemblies in the mammalian cell • Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes • NADH binding site in the matrix side • Non-covalently bound flavin mononucleotide (FMN) accepts two electrons from NADH • Several iron-sulfur centers pass one electron at the time toward the ubiquinone binding site
  • 24. NADH:Ubiquinone Oxidoreducase is a Proton Pump • Transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P) • Experiments suggest that about four protons are transported per one NADH NADH + Q + 5H+ N = NAD+ + QH2 + 4 H+ P • Reduced coenzyme Q picks up two protons • Despite 50 years of study, it is still unknown how the four other protons are transported across the membrane
  • 25. Iron-Sulfur Centers • Found in several proteins of electron transport chain, including NADH:ubiquinone oxidoreductase • Transfers one electron at a time
  • 26. Succinate Dehydrogenase a.k.a. Complex II • FAD accepts two electrons from succinate • Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone that becomes reduced QH2
  • 27. • The cytochromes are proteins with characteristic strong absorption of visible light, due to their iron-containing heme prosthetic groups. Mitochondria contain three classes of cytochromes, designated a, b, and c, which are distinguished by differences in their light- absorption spectra. • Each type of cytochrome in its reduced (Fe2) state has three absorption bands in the visible range
  • 28. Cytochrome bc1 Complex a.k.a. Complex III • Uses two electrons from QH2 to reduce two molecules of cytochrome c
  • 29. The Q Cycle • 4 H+ / 2 e- that reach CytC • 2 H+ from QH2 • 2 H+ from the matrix
  • 30. Cytochrome c • Cytochrome c is a soluble heme-containing protein in the intermembrane space • Heme iron can be either ferrous(Fe3+, oxidized) or ferric(Fe2+, reduced) • Cytochrome c carries a single electron from the cytochrome bc1 complex to cytochrome oxidase
  • 31. Cytochrome c Absorbs Visible Light • Intense Soret band near 400 nm absorbs blue light and gives cytochrome c an intense red color • Cytochromes are sometimes named by the position of their longest- wavelength peak
  • 32. Cytochrome Oxidase a.k.a. Complex IV • Mammalian cytochrome oxidase is a membrane protein with 13 subunits • Contains two heme groups • Contains copper ions – Two ions (CuA) form a binuclear center – Another ion (CuB) bonded to heme forms Fe-Cu center
  • 33. Cytochrome C Oxidase (complex IV) Transport
  • 34. Structure of the Cytochrome C Oxidase Monomer • The heme groups are shown in blue and red and copper sites in green • The catalytic core consists of I yellow, II blue, III pink • The entire complex consists of 13 subunits
  • 35. A proposed reaction cycle for the four-electron reduction of O2 by cytochrome oxidase (at the Heme a3-CuB center)
  • 36. Structure of Beef Heart Cytochrome Oxidase The protein is a dimer of two 13 monomers 3 dimensional structure of beef heart cytochrome oxidase at 2.8 angstrom resolution
  • 37. The order of the many electron carriers on the respiratory chain have been elucidated via various studies • Measurement of the standard reduction potential (E`0)): Electrons tend to transfer from low E`0 carriers to high E`0 carriers (but may deviate from this in real cells). • Oxidation kinetics studies: Full reduction followed by sudden O2 introduction; earlier oxidation, closer to the end of the respiratory chain; using rapid and sensitive spectrophotometric techniques to follow the oxidation of the cytochromes, which have different wavelength of maximal absorption).
  • 38. Electron carriers may have an order of increasing E`0
  • 39. • the standard reduction potentials of the individual electron carriers have been determined experimentally . We would expect the carriers to function in order of increasing reduction potential, because electrons tend to flow spontaneously from carriers of lower E to carriers of higher E. • The order of carriers deduced by this method is NADH → Q → cytochrome b → cytochrome c1 → cytochrome c → cytochrome a → cytochrome a3 → O2.
  • 40. • Effects of various specific inhibitors: those before the blocked step should be reduced and those after be oxidized. • Isolation and characterization of each of the multiprotein complexes: specific electron donors and acceptors can be determined for portions of the chain.
  • 41. Various inhibitors generate various patterns of reduced/oxidized carriers Reduced Oxidized Reduced Oxidized Reduced
  • 42. Oxidative Phosphorylation (0n inner membrane of mitochondria)
  • 43. Electron transfer to O2 was found to be coupled to ATP synthesis from ADP + Pi in isolated mitochondria • ATP would not be synthesized when only ADP and Pi are added in isolated mitochondria suspensions. • O2 consumption, an indication of electron flow, was detected when a reductant (e.g., succinate) is added, accompanied by an increase of ATP synthesis. • Both O2 consumption and ATP synthesis were suppressed when inhibitors of respiratory chain (e.g., cyanide, CO, or antimycin A) was added. • ATP synthesis depends on the occurrence of electron flow in mitochondria.
  • 44. • O2 consumption (thus electron flow) was neither observed if ADP was not added to the suspension, although a reductant is provided! • The O2 consumption was also not observed in the presence of inhibitors of ATP synthase (e.g., oligomycin or venturicidin). • Electron flow also depends on ATP synthesis!
  • 45. Electron transfer was found to be obligatorily coupled to ATP Synthesis in isolated mitochondria suspensions: neither occurs without the other.
  • 46. 3 D Model of ATP Synthase: An Electrical Mechano-Chemical Molecular Complex • The Fo portion is composed of integral transmembranous proteins a, b and 9-14 copies of c which forms a ring-like structure in the plane of the membrane. • The F1 head piece is composed of a hexagonal array of alternating  and  subunits, a central  protein with a helical coil that associates with  and  proteins and extends into the c protein ring in the Fo.
  • 47. Atomic Force Microscopy of C-subunit Ring Structures Isolated from Chloroplast ATP Synthase and Inserted Into Liposomes
  • 48. c ring & a subunit structure •each c subunit has 2 membrane- spanning a helices – midway along 1 helix: asp – COOH↔COO– •a subunit has 2 half-channels H+ path •H+ from cytosol diffuses via half- channel to asp on c ring subunit (c1) •this subunit can now move to interface membrane, allowing c ring to rotate •c9 now interfaces matrix half-channel, allowing H+ to diffuse into matrix c ring subunit a H+ path through membrane c1 c9 matrix half- channel cytosolic half-channel asp subunit ac subunit
  • 49. cannot rotate in either direction can rotate clockwise matrix H+ flow drives rotation of c ring
  • 50. Binding-change mechanism of ATP synthesis • Rotation of gama subunit drives release of tightly bound ATP • 3 active sites cycle through 3 structural states: O, open; L, loose-binding; T, tight-binding • At T site, ADP + Pi  ATP, but ATP can’t dissociate • G rotation causes T  O, L  T, O  L • As a result of the TO structural change, ATP can now dissociate from what is now an O site. T O ATP ADP + Pi ATP 120° rotation of  (counterclockwise) T TO O L L 1 1 2 2 3 3
  • 51. Synthesis of ATP: Rotary Catalysis • ATP is synthesized by coupling the energy liberated during proton translocation through the FoF1 to a motive force that rotates the C ring structure and the attached  subunit. • -subunits contain the catalytic sites of ATP synthesis. 120 degree units of rotation of the  protein around the stationary / hexagonal array results in altered associations of the  protein with the  protein forming the L, T and O states for the 3 β- subunits. ATP is produced in the T state where the ∆G = ~ 0. • Each rotation of 360 degrees of the γ subunit results in 3 ATP, one for each β-subunit. The model shows the rotation as arbitrarily clockwise. ∆G = ~ 0
  • 52. Nature 386, 299 - 302 (20 March 1997); doi:10.1038/386299a0 Direct observation of the rotation of F1-ATPase HIROYUKI NOJI*, RYOHEI YASUDA†, MASASUKE YOSHIDA* & KAZUHIKO KINOSITA JR† †Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223, Japan
  • 53. Transport across inner mitochondrial membrane • p also drives flow of substances across inner membrane • Transported by specific carrier proteins • Cotransport: coupled transport of 2 substances –Symport: both move in same direction CH3CCOO– + H+ O HPO4= + H+ –Antiport: each moves in opposite direction ADP-ATP exchange
  • 54. Active Transport of ATP, ADP & Pi • Adenine Nucleotide Translocase – Antiporter – (ATP4- matrix  ADP3- inter membrane) • Phosphate trans locase – Symporter – {Pi- , H+} inter membrane => {Pi- , H+} matrix
  • 55. Summary of ATP synthesis & translocation of ATP,ADP & Pi
  • 56. Energy of Light is Used to Synthesize ATP in Photosynthetic Organisms • Light causes charge separation between a pair chlorophyll molecules • Energy of the oxidized and reduced chlorophyll molecules is used drive synthesis of ATP • Water is the source of electrons that are passed via a chain of transporters to the ultimate electron acceptor, NADP+ • Oxygen is the byproduct of water oxidation
  • 57. Various Pigments Harvest the Light Energy The energy is transferred to the photosynthetic reaction center
  • 58. Light-Induced Redox Reactions and Electron Transfer Cause Acidification of Lumen The proton-motive force across the thylakoid membrane drives the synthesis of ATP
  • 59. Flow of Protons: Mitochondria, Chloroplasts, Bacteria • Mitochondria and chloroplasts arose endosymbionts - entrapped bacteria • Bacterial cytosol became mitochondrial matrix and chloroplast stroma
  • 60. Photophosphorylation (on thylakoid of chloroplasts)
  • 61. References • Lehninger Principles of Biochemistry, 5th Edition- © 2008 W.H Freeman and company. • Fundamentals of Biochemistry- a text book , H.P. Gajera, S.V. Patel, B.A. Golakiya. • Fundamentals of Biochemistry- J.L. Jain, Sunjay Jain, Nitin Jain.