stryer_biochem10e_lectureslides_ch18_Zhang (1).pptx

Chapter 18 Oxidative Phosphorylation © 2023 W. H. Freeman and Company Ch.18 Learning Goals By the end of this chapter, you should be able to: 1. Describe the key components of the electrontransport chain and how they are arranged. 2. Explain the benefits of having the electron-transport chain loc...

Chapter 18 Oxidative Phosphorylation © 2023 W. H. Freeman and Company Ch.18 Learning Goals By the end of this chapter, you should be able to: 1. Describe the key components of the electrontransport chain and how they are arranged. 2. Explain the benefits of having the electron-transport chain located in a membrane. 3. Describe how the proton-motive force is converted into ATP. 4. Identify the ultimate determinant of the rate of cellular respiration. Ch.18 Outline • 18.1 Cellular Respiration Drives ATP Formation by Transferring Electrons to Molecular Oxygen • 18.2 Oxidative Phosphorylation Depends on Electron Transfer • 18.3 The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle • 18.4 A Proton Gradient Powers the Synthesis of ATP • 18.5 Many Shuttles Allow Movement Across Mitochondrial Membranes • 18.6 The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP • 18.7 Proton Gradients Generated by Respiratory Chains Drive Many Biochemical Processes Section 18.1 Cellular Respiration Drives ATP Formation by Transferring Electrons to Molecular Oxygen • respiratory chain (electron transport chain) = four large protein complexes that are embedded in the inner mitochondrial membrane • oxidative phosphorylation = set of electron-transfer reactions that captures the energy of high-energy electrons from NADH and FADH2 – takes place in the electron-transport chain – ultimately generates ATP and reduces oxygen to water Coupling of Electron Carrier Oxidation and ADP Phosphorylation • The flow of electrons from reduced carriers such as NADH is highly exergonic. NADH  1 2 O2  H  H2O  NAD   G  220.1 kJ mol 1  52.6 kcal mol  1  • Complexes of the electron-transport chain use released energy to pump protons out of the mitochondrial matrix. – generates a pH gradient and a transmembrane electron potential that creates a proton-motive force that is used to power the synthesis of ATP ADP  Pi  H  ATP  H2O  G  30.5 kJ mol 1 7.3 kcal mol  1  Oxidative Phosphorylation Is Fundamentally the Combination of Two Processes Respiration • respiration (cellular respiration) = the generation of hightransfer-potential electrons by the citric acid cycle, their flow through the respiratory chain, and the accompanying synthesis of ATP Eukaryotic Oxidative Phosphorylation Takes Place in Mitochondria • The citric acid cycle, the electron-transport chain, and ATP synthesis occur in the mitochondria. • Mitochondria have two membranes: – an outer membrane – an extensive, highly folded inner membrane • intermembrane space (IM space) = compartment between the outer and inner membranes • matrix = compartment bounded by the inner membrane Mitochondria Are Bound by a Double Membrane, Which Creates Two Distinct Internal Compartments • Most citric acid cycle and fatty acid oxidation reactions take place in the mitochondrial matrix. • Oxidative phosphorylation takes place in the inner mitochondrial membrane. The Membranes of Mitochondria • The outer mitochondrial membrane is permeable to most small molecules and ions. – because of the presence of mitochondrial porin (voltagedependent anion channel) • The inner membrane is folded into internal ridges and tube-like structures called cristae that increase the surface areas to create more sites for oxidative phosphorylation. – impermeable to most ions and polar molecules – transporters shuttle metabolites across the membrane – has two sides: the matrix side and the cytoplasmic side Mitochondria Are the Result of an Endosymbiotic Event (1 of 2) • Mitochondria are semiautonomous organelles that live in an endosymbiotic relationship with the host cell. – contain their own DNA • the endosymbiotic theory = hypothesizes that an endosymbiotic event occurred in the evolution of eukaryotic organisms in which a free-living organism capable of oxidative phosphorylation was engulfed by another cell • Sequence data suggest that all mitochondria are descendants of an ancestor of Rickettsia prowazekii as the result of a single endosymbiotic event. Mitochondria Are the Result of an Endosymbiotic Event (2 of 2) • The most bacteria-like mitochondrial genome is that of the protozoan Reclinomonas americana. • The R. americana genome encodes less than 2% of the protein-encoding genes of E. coli. • All mitochondrial genomes have approximately the same 2% of bacterial genes. – suggests that an endosymbiotic event occurred just once in evolution Which statement is true of cellular respiration? (1 of 2) a. Transmembrane proton fluxes couple the citric acid cycle with the flow of electrons from NADH and FADH2 through the electron-transport chain. b. Electrons from NADH and FADH2 flow through the electron-transport chain to reduce water to oxygen. c. Some components of the electron-transport chain pump protons from the intermembrane space to the matrix. d. The matrix is the site of most of the reactions of the citric acid cycle, whereas oxidative phosphorylation takes place in the inner mitochondrial membrane. e. The flow of electrons from NADH is highly endergonic. © Macmillan Learning, 2023 Which statement is true of cellular respiration? (2 of 2) a. Transmembrane proton fluxes couple the citric acid cycle with the flow of electrons from NADH and FADH2 through the electron-transport chain. b. Electrons from NADH and FADH2 flow through the electron-transport chain to reduce water to oxygen. c. Some components of the electron-transport chain pump protons from the intermembrane space to the matrix. *d. The matrix is the site of most of the reactions of the citric acid cycle, whereas oxidative phosphorylation takes place in the inner mitochondrial membrane. e. The flow of electrons from NADH is highly endergonic. © Macmillan Learning, 2023 Section 18.2 Oxidative Phosphorylation Depends on Electron Transfer • In oxidative phosphorylation, the electron-transfer potential of NADH or FADH2 is converted into the phosphoryl-transfer potential of ATP. • phosphoryl-transfer potential = the ∆G°′ for the hydrolysis of the activated phosphoryl compound • electron-transfer potential = E′0, the reduction potential (redox potential, oxidation–reduction potential) – measures a molecule's tendency to donate or accept electrons The Electron-Transfer Potential of an Electron Is Measured as Redox Potential • redox couple = a substance that can exist in an oxidized form X and a reduced form X− – designated X : X− – reduction potential can be determined by measuring the electromotive force generated by an apparatus called a sample half-cell connected to a standard reference half-cell Redox Potential Can Be Precisely Measured • The agar bridge allows ions to move from one half-cell to the other. • Electrons flow from one half-cell to the other through the wire connecting the two half-cells to the voltmeter. Reactions in the Half-Cells • if the reaction proceeds in the direction X  H  X  1 2 H2 • the reactions in the half-cells must be X  X  e H  e   1 2 H2 • Electrons flow from the sample half-cell to the standard reference half-cell. • The sample-cell electrode is taken to be negative with respect to the standard-cell electrode. Reductants and Oxidants • reductant (reducing agent) = the donor of electrons – X− in the example • oxidant (oxidizing agent) = acceptor of electrons – H+ in the example X  H  X  1 2 H2 Reduction Potential • Negative E′0 means that the oxidized form of a substance has lower affinity for electrons than does H2. – example: NADH is a strong reducing agent and has a negative E′0 • Positive E′0 means that the oxidized form of a substance has higher affinity for electrons than does H2. – example: O2 is a strong oxidizing agent and has a positive E′0 • The driving force of oxidative phosphorylation is the E′0 of NADH or FADH2 relative to that of O2. Standard Reduction Potentials of Some Reactions TABLE 18.1 Standard reduction potentials of some reactions Oxidant Reductant n E0  V Succinate +CO2 α-Ketoglutarate 2 –0.67 Acetate Acetaldehyde 2 –0.60 Ferredoxin (oxidized) Ferredoxin (reduced) 1 –0.43 2 H+ H2 2 –0.42 NAD+ NADH + H+ 2 –0.32 NADP+ NADPH + H+ 2 –0.32 Lipoate (oxidized) Lipoate (reduced) 2 –0.29 Glutathione (oxidized) Glutathione (reduced) 2 –0.23 FAD FADH2 2 –0.22 Acetaldehyde Ethanol 2 –0.20 Pyruvate Lactate 2 –0.19 2H H2 2 0.001 Fumarate Succinate 2 –0.03 Cytochrome b(+3) Cytochrome b(+2) 1 +0.07 Dehydroascorbate Ascorbate 2 +0.08 Ubiquinone (oxidized) Ubiquinone (reduced) 2 +0.10 Cytochrome c(+3) Cytochrome c(+2) 1 +0.22 Fe (+3) Fe (+2) 1 +0.77 H2O 2 +0.82 + ½O2 + 2 H + Note: E0 is the standard oxidation–reduction potential (pH 7,25°C), and n is the number of electrons transferred. 1 E0 refers to the partial reaction written as Oxidant + e – → reductant. Standard oxidation – reduction potential at pH = 0. Compare with E0  0.42 at pH = 7. The Relationship Between ∆G°′ and ∆E′0 • Standard free-energy change ∆G°′ is related to the change in reduction potential ∆E′0 by G   nF E0 (1) where n is the number of electrons transferred, and F is a proportionality constant called the Faraday constant (96.48 kJ mol−1 V−1) What is n in the following reaction? ½O2 + NADH + H+ → H2O + NAD+ (1 of 2) © Macmillan Learning, 2023 What is n in the following reaction? ½O2 + NADH + H+ → H2O + NAD+ (2 of 2) 2 © Macmillan Learning, 2023 Electron Flow from NADH to Molecular Oxygen Powers the Formation of a Proton Gradient • to calculate ΔG°for the reduction O2 with NADH, consider the pertinent half-reactions 1  2 H  2 e  H2O 2 O2 NAD  H  2 e   NADH E0 0.82 V (A) E0  0.32 V (B) • combining the two half-reactions yields 1 2 O2  NADH  H  H2O  NAD  (C) • the standard free energy for this reaction is –220.1 kJ mol-1 ΔG°= (–2 x 96.48 kJ mol-1 V-1 x +0.82 V) + (–2 x 96.48 kJ mol-1 V-1 x +0.32 V) Quantification of Energy Associated with a Proton Gradient • The energy associated with a proton gradient can be quantified by: ΔG = RT ln (c2 /c1 ) + ZFΔV (2) where c1 is the concentration of the protons on one side of the membrane and c2 is the concentration of protons on the side of the gradient to which the protons are moving, Z is the electrical charge of the proton, ΔV is the voltage potential across the membrane, R is the gas constant, and T is the temperature in kelvin Section 18.3 The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle • Electrons flow from NADH to O2 through three protein complexes embedded in the inner mitochondria membrane: – NADH-Q oxidoreductase (Complex I) – Q-cytochrome c oxidoreductase (Complex III) – cytochrome c oxidase (Complex IV) • Electron flow through Complexes I, III, and IV is highly exergonic and powers generation of a proton gradient. • Complexes I, III, and IV are proton pumps. Complex II • Succinate Q-reductase (Complex II) contains succinate dehydrogenase from the citric acid cycle. – Electrons from this FADH2 enter the electron-transport chain at Q-cytochrome c oxidoreductase. – It does not pump protons. Components of the ElectronTransport Chain Allow the Flow of Electrons to Molecular Oxygen • Complexes I, III, and IV appear to be associated in a supramolecular complex. – facilitates the rapid transfer of substrate – prevents the release of reaction intermediates Components of the Mitochondrial Electron-Transport Chain TABLE 18.2 Components of the mitochondrial electron-transport chain Oxidant or reductant Enzyme complex Prosthetic group Matrix side > 900 FMN Fe-S NADH Q Succinate-Q reductase 140 FAD Fe-S Succinate Q Q-cytochrome c oxidoreductase 250 Heme bH Heme bL Heme c1 Fe-S Cytochrome c oxidase 160 Heme a Heme a3 CuA and CuB NADH-Q oxidoreductase Mass (kDa) Membrane core Q Intermembrane side Cytochrome c Cytochrome c Information from: J. W. DePierre and L. Ernster, Annu. Rev. Biochem. 46:215, 1977; Y. Hatefi, Annu. Rev. Biochem. 54:1015, 1985; and J. E. Walker, Q. Rev. Biophys. 25:253, 1992. Coenzyme Q • coenzyme Q (Q or ubiquinone) = hydrophobic quinone that diffuses rapidly within the inner mitochondrial membrane – derived from isoprene – can exist in several oxidation states – couple electron-transfer reactions to proton binding and release • Coenzyme Q functions as an electron carrier. – The reduced form carries electrons from Complex I to Complex III. – It transfers electrons from FADH2 from the citric acid cycle to Complex III. Quinones Can Have Three Different Oxidation States, Two of Which Are Stable Cytochrome c • cytochromes = electron-transferring proteins that contain a heme prosthetic group • cytochrome c = small soluble protein that is loosely associated with the inner mitochondrial membrane – shuttles electrons from Complex III to Complex IV – present in all organisms with mitochondrial respiratory chains – highly conserved across species – cytochrome c from any eukaryotic species will react in vitro with the cytochrome c oxidase from any other species The Three-Dimensional Structure of Cytochrome c Is Remarkably Conserved Among Distantly Related Species Iron–Sulfur Clusters Are Common Components of the ElectronTransport Chain • iron–sulfur clusters = play a critical role in reduction reactions in biological systems – exist within iron–sulfur proteins (nonheme iron proteins) – three kinds are common in biological systems: a single iron ion, 2Fe-2S, and 4Fe-4S – iron ions cycle between Fe2+ (reduced) and Fe3+ (oxidized) states – undergo oxidation–reduction reactions without releasing or binding protons The Protein Frataxin • frataxin = a small mitochondrial protein that is crucial for the synthesis of Fe-S clusters • Friedreich's ataxia = a disease of varying severity that affects the central and peripheral nervous system as well as the heart and skeletal system – caused by mutations in frataxin Three Different Types of Iron–Sulfur Clusters Are Commonly Found in Biological Systems The High-Potential Electrons of NADH Enter the Respiratory Chain at NADH-Q Oxidoreductase • NADH-Q oxidoreductase (Complex I) = proton pump that serves as the entry point for electrons from NADH – encoded by genes in the mitochondria and nucleus – L-shaped, with a horizonal arm in the membrane and a vertical arm projecting into the matrix • Complex I catalyzes the reaction + + NADH  Q  5 Hmatrix  NAD  QH2  4Hintermembrane space Flavins Have Two Oxidation States • NADH binds and the transfer of its two high-potential electrons to the flavin mononucleotide (FMN) prosthetic group of Complex I – yields the reduced form, FMNH2 – electrons are then passed to a series of iron–sulfur proteins clusters in Complex I The Half-Channels of Complex I • Membrane-embedded part of Complex I has four proton half-channels containing of vertical helices. – One set is exposed to the matrix and the other set to the intermembrane space. – Vertical helices are linked on the matrix side by a long horizonal helix (HL). – The intermembrane space half-channels are joined by βhairpin-helix connecting elements (βH). Electron Transfer Through NADH-Q Oxidoreductase Is Coupled to Proton Transfer Reactions • Q chamber = enclosed site where Q accepts electrons from NADH Structural Cooperation Pumps Protons Out of the Matrix • Q accepts two electrons from NADH, generating Q2−. • Q2− negative charge causes a conformational change in LH and βH elements, leading to a change in the structures of the connected vertical helices that change the pKa of the amino acids • H+ from the matrix binds to amino acids, dissociates into a water-lined channel, and enters the intermembrane space. • The flow of two electrons from NADH to coenzyme Q through Complex I leads to the pumping of 4 H+ out of the matrix of the mitochondrion. The Reduction of Q2− to QH2 • Q2− takes up two protons from the matrix as it is reduced to QH2. – contributes to the formation of the proton-motive force • QH2 subsequently leaves the enzyme for the Q pool, allowing another reaction cycle to occur. Ubiquinol Is the Entry Point for Electrons from FADH2 of Flavoproteins • succinate-Q reductase (Complex II) = integral membrane protein complex of the inner mitochondrial membrane – contains succinate dehydrogenase – does not pump protons, resulting in less ATP being formed in the oxidation of FADH2 than NADH • Electrons from FADH2 (generated in the citric acid cycle) are transferred to Fe-S centers and then to Q to form QH2. Electrons Flow from Ubiquinol to Cytochrome c Through Q-Cytochrome c Oxidoreductase • Electrons from QH2 are passed to cytochrome c (Cyt c) by Q-cytochrome c oxidoreductase (Complex III). – leads to the net transport of 2 H+ to the intermembrane space + + QH2  2 Cyt cox  2 Hmatrix  Q  2 Cyt cred  4Hintermembrane space • Complex III contains: – two types of cytochromes named b and c1 – four prosthetic groups: three hemes and a 2F3-2S cluster Q-cytochrome c Oxidoreductase Is a Homodimer • Heme prosthetic group in the cytochromes is iron-protoporphyrin IX. • The iron ion of the cytochrome alternates between Fe2+ and Fe3+. • The cytochromes contain three hemes: – two heme bL (L for low affinity) – one heme bH (H for high affinity) The Q Cycle Funnels Electrons from a Two-Electron Carrier to a One-Electron Carrier While Pumping Protons • QH2 carries two electrons, whereas the Complex III electron acceptor, cytochrome c, carries only one electron. • Q cycle = the mechanism for coupling electron transfer from QH2 to cytochrome c to transmembrane proton transport • In one cycle, four protons are pumped out of the mitochondrial matrix and two are removed from the matrix + 2QH2  Q  2Cyt cox  2 H+matrix  2 Q  QH2  2Cyt cred  4Hintermembrane space The First Half of the Q Cycle • One reduced (QH2) and one oxidized (Q) molecule of Q bind to the Qo and Qi sites, respectively. • Bound QH2 passes its two electrons through the complex: – One electron flows to the 2Fe-2S cluster, then to cytochrome c1, and finally to oxidized cytochrome c, reducing it. – The other electron passes through two heme groups of cytochrome b to the bound Q, reducing it to a dangerous semiquinone radical anion (Q⋅− ) that stays tightly bound. The Second Half of the Q Cycle • A second QH2 binds to the Qo site, and its electrons pass through the complex as in the first half. • On addition of the second electron, the radical accepts two protons from the matrix and forms QH2. The Q Cycle Allows the Safe Transfer of Electrons from QH2 to Cytochrome c With each full Q cycle, how many oxidized Q and reduced QH2 leave? (1 of 2) © Macmillan Learning, 2023 With each full Q cycle, how many oxidized Q and reduced QH2 leave? (2 of 2) two Q and one QH2 © Macmillan Learning, 2023 Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen to Water • cytochrome c oxidase (Complex IV) = catalyzes the transfer of four electrons from four reduced molecules of cytochrome c to O2 • Four H+ are used to reduce O2 to H2O. • Four H+ are pumped into the intermembrane space. • ∆G°′ for this reaction is −231.8 kJ mol−1. – captured in the form of a proton gradient for ATP synthesis + + 4Cyt cred  8 Hmatrix  O2  4Cyt cox  2H2O  4Hintermembrane space Structure of Bovine Cytochrome c Oxidase Copper Centers of Cytochrome c Oxidase • Cytochrome c oxidase consists of: – 13 subunits. – two heme A groups and three Cu ions, arranged as two Cu centers: CuA/CuA and CuB. • CuA/CuA contains two copper ions linked by two bridging Cys residues. – initially accepts electrons from reduced cytochrome c • CuB is bonded to three His residues, one of which is covalently linked to Tyr. Heme A Molecules of Cytochrome c Oxidase • Cytochrome c oxidase contains heme a and heme a3. – have different redox potentials due to different environments within the enzyme – heme a3 and CuB for the active center at which O2 is reduced to H2O Four Molecules of Cytochrome c Bind Consecutively and Transfer an Electron to Reduce O2 to H2O (1 of 2) • Step 1: electrons from two reduced cytochrome c molecules flow to CuA/CuA, to heme a, to heme a3, to CuB – one stops at heme a3 – one stops at CuB • Step 2: reduced heme a3 and CuB bind O2, forming a peroxide bridge between them Four Molecules of Cytochrome c Bind Consecutively and Transfer an Electron to Reduce O2 to H2O (2 of 2) • Step 3: electrons from two more cytochrome c molecules and H+ from the matrix bind to each oxygen, cleaving the peroxide bridge • Step 4: reactions with two more H+ releases two molecules of H2O and resets the enzyme The Mechanism of Cytochrome c Oxidase Prevents Early Oxygen Release Two Metals Form a Peroxide Bridge in Cytochrome c Oxidase Proton Transport by Cytochrome c Oxidase Has Two Components • Four chemical protons reduce O2 to two H2O. • Cytochrome c oxidase uses free energy from this reduction to pump 4 H+ from the matrix into the intermembrane space. Electrons Flow via Two Pathways Through the Electron-Transport Chain Most of the Electron-Transport Chain Is Organized into a Larger Complex Called the Respirasome • respirasome = a massive complex in humans consisting of two copies of Complex I, Complex III, and Complex IV – structure allows for Complex II to associate in a gap between Complexes I and IV – enhances efficiency Toxic Derivatives of Molecular Oxygen Such as Superoxide Radicals Are Scavenged by Protective Enzymes • Partial reduction of O2 generates highly reactive oxygen derivatives called reactive oxygen species (ROS). • ROS are implicated in aging and a growing list of diseases. • ROS include superoxide ion, peroxide ion, and hydroxyl radical (OH). • Cytochrome c oxidase does not release ROS by holding O2 tightly between Fe and Cu ions. e O2    O2 . Superoxide ion e   O2 2 Peroxide Pathological Conditions That May Entail Free-Radical Injury TABLE 18.3 Pathological conditions that may entail free-radical injury Atherogenesis Emphysema; bronchitis Parkinson's disease Duchenne muscular dystrophy Cervical cancer Alcoholic liver disease Diabetes Acute renal failure Down syndrome Retrolental fibroplasia Cerebrovascular disorders Ischemia; reperfusion injury Information from M. Lieberman and A. D. Marks, Basic Medical Biochemistry: A Clinical Approach, 4th ed. (Lippincott, Williams & Wilkins, 2012), p. 437. Superoxide Dismutase • superoxide dismutase (SOD) = enzyme that scavenges superoxide radicals by catalyzing the conversion of two radicals into hydrogen peroxide and molecular oxygen • Eukaryotes contain two forms of SOD: – a manganese-containing version located in mitochondria – a copper-and-zinc-dependent cytoplasmic form • exercise is associated with increased SOD expression • functions near diffusion-limited rate The Superoxide Dismutase Mechanism Has Two Phases Catalase • catalase = a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen • functions near diffusion-limited rate Superoxide dismutase: (1 of 2) a. reacts with superoxide and two protons when in its oxidized form. b. contains manganese in the cytoplasmic form. c. is synthesized in higher levels in response to elevated aerobic metabolism. d. catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. e. catalyzes the conversion of one superoxide radical and two hydrogen peroxide to two water and one oxygen. © Macmillan Learning, 2023 Superoxide dismutase: (2 of 2) a. reacts with superoxide and two protons when in its oxidized form. b. contains manganese in the cytoplasmic form. *c. is synthesized in higher levels in response to elevated aerobic metabolism. d. catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. e. catalyzes the conversion of one superoxide radical and two hydrogen peroxide to two water and one oxygen. © Macmillan Learning, 2023 Electrons Can Be Transferred Between Groups That Are Not in Contact • In a protein environment, the rate of electron transfer decreases by a factor of 10 for every 1.7 Å distance between electron donor and acceptor. • For groups in contact, electron-transfer reaction rates are ~ 1013 s−1. • For electron-transport chain proteins, electron-carrying groups are typically separated by 15 Å beyond their van der Waals contact distance. – rates are ~104 s−1 Proteins Dramatically Improve the Rate of Electron Transfer Between Atoms at a Distance Section 18.4 A Proton Gradient Powers the Synthesis of ATP • The flow of NADH to O2 is an exergonic process: • The synthesis of ATP is an endergonic process: • ATP synthase (Complex V) = a molecular assembly in the inner mitochondrial membrane that carries out the synthesis of ATP The Chemiosmotic Hypothesis Suggested That ATP Formation Is Powered by a Proton Gradient • chemiosmotic hypothesis = proposes that electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane The Proton-Motive Force • proton-motive force = the energy-rich unequal distribution of protons across a membrane – consists of a chemical gradient and a charge gradient – powers the synthesis of ATP Proton-motive force  p   chemical gradient  pH   charge gradient    The Chemiosmotic Hypothesis Is Supported by Experiments Using Bacterial Proton Pumps • When the vesicles were exposed to light, ATP was formed. – showed the respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force ATP Synthase Is Composed of a Proton-Conducting Unit and a Catalytic Unit • ATP synthase, resembling a ball on a stick, is made up of two components: – The F0 (stick) component is embedded in the inner mitochondrial membrane and contains the proton channel. – The F1 (ball) component protrudes into the mitochondrial matrix and contains the catalytic activity. The Structure of ATP Synthase Reveals a Complex Molecular Rotational Motor The F1 Subunit • The F1 subunit consists of five types of polypeptide chains: α3, β3, γ , δ, and ε. • α and β subunits are arranged in a hexameric ring. – Both bind nucleotides. – Only the β subunits are catalytically active. • A central stalk consists of the γ and ε proteins. • The γ subunit breaks the symmetry of the α3β3 hexamer. – Each β subunit is distinct by virtue of its interaction with a different, asymmetrical, face of γ. The F0 Subunit • The F0 subunit consists of a ring comprising from 8 to 14 c subunits that are embedded in the membrane. • A single a subunit binds to the outside of the ring. • The F0 and F1 subunits are connected in two ways: – the central γε stalk – an exterior column consisting of one a subunit, two b subunits, and the δ subunit Mitochondria ATP Synthase Forms Homodimers • stabilizes the individual enzymes to the rotational forces required for catalysis • facilitates the curvature of the inner mitochondrial membrane ATP Synthase Assists in the Formation of Cristae • Cristae formation allows proton pumps to localize the proton gradient in the vicinity of the synthases, which are located at the tips of the cristae. – enhances efficiency of ATP synthesis Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound ATP via the Binding-Change Mechanism • ATP synthase catalyzes the formation of ATP from ADP and Pi • Actual substrates are ATP and ADP complexed with Mg2+. • A terminal oxygen atom of ADP attacks the phosphorus atom of Pi. – form a pentacovalent intermediate that dissociates into ATP and H2O ATP-Synthesis Is Catalyzed by ATP Synthase ATP Forms Without a Proton-Motive Force But Is Not Released • Isotopic-exchange experiments revealed enzyme-bound ATP forms readily in the absence of a proton-motive force. • ATP does not leave the catalytic site unless protons flow through the enzyme. Proton Flow Through the ATP Synthase Allows Release of the Newly Synthesized ATP • The F1 subunit of the ATPase contains three β subunits – There are three active sites on the enzyme. • The binding-change mechanism states a β subunit can perform three sequential steps in ATP synthesis by changing conformation: – Step 1: ADP and Pi binding – Step 2: ATP synthesis – Step 3: ATP release ATP Synthase Nucleotide-Binding Sites Cycle Through Distinct Conformational States The Three Conformational States of the β Subunits • The L (loose) conformation binds ADP and Pi. – cannot release bound nucleotides • The T (tight) conformation binds ATP with great affinity. – converts bound ADP and Pi to ATP – cannot release bound nucleotides • The O (open) conformation can bind or release adenine nucleotides. The Binding-Change Mechanism Involves the Cycling of All Three β Subunits Through Three Conformations • The rotation of the γ subunit drives the interconversion of these three forms. • Each subunit progresses from T to O to L form. • No two subunits are in the same conformational form. Rotational Catalysis Is the World's Smallest Molecular Motor • Cloned α3β3γ subunits were attached to a glass slide. – The γ subunit was linked to a fluorescently labeled actin filament to provide a segment that could be observed under a fluorescence microscope. • ATP addition caused the actin filament to rotate in a counterclockwise direction. – visually confirmed that the γ subunit was rotating, driven by the hydrolysis of ATP • The hydrolysis of a single ATP powered the rotation of the γ subunit 120°. The ATP-Driven Rotation in ATP Synthase Has Been Directly Observed Proton Flow Around the c Ring Powers ATP Synthesis • Proton flow occurs through the Fo component of the ATP synthase. • The stationary a subunit abuts the membrane-spanning ring form by c subunits. • a subunit contains two hydrophilic half-channels, each of which interacts with one c subunit. – One opens to the intermembrane space. – One opens to the matrix. The Structure of the c Subunit • the membrane-spanning ring formed by 8 to 14 c subunits – vertebrates have 8 units • Each polypeptide chain of the c subunit forms a pair of α helices that span the membrane. • A glutamic acid (or aspartic acid) residue is found in the middle of one of the helices. – When unprotonated, the c subunit will not move into the membrane. – When protonated, the c subunit moves into the membrane as the ring rotates by one c subunit. Components of the ProtonConducting Unit of ATP Synthase The Rotation of the c Ring • Step 1: The proton enters the half-channel from the proton-rich intermembrane space. • Step 2: The proton binds to a Glu or Asp residue in a c subunit. • Step 3: The c ring rotates clockwise, one c subunit at a time. • Step 4: The proton exits the half-channel to the matrix following a complete rotation of the c ring. The Rotation of the c Ring, Continued • Movement of protons through the half-channels from the high proton concentration to the low proton concentration powers the rotation of the c ring. • The rotation of the c rings powers the movement of the γ subunit, which in turn alters the conformation of the β subunits. Proton Motion Across the Membrane Drives Rotation of the c Ring Protons Must Travel Around the c Ring to Cross the Membrane • Each 360-degree rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. An Overview of Oxidative Phosphorylation Shows the Spatial Relationship of the Components ATP Synthase and G Proteins Have Several Common Features • α and β subunits of ATP synthase and G proteins are members of the P-loop NTPase family of proteins. – signaling properties depend on their ability to bind NTPs and NDPs – do not exchange nucleotides unless stimulated by interaction with other proteins The number of c subunits in the c ring determines the number of protons that must be transported to generate a molecule of ATP. If there are 14 c subunits, how many protons are required to generate each ATP molecule? (1 of 2) © Macmillan Learning, 2023 The number of c subunits in the c ring determines the number of protons that must be transported to generate a molecule of ATP. If there are 14 c subunits, how many protons are required to generate each ATP molecule? (2 of 2) 14/3 = 4.67 protons © Macmillan Learning, 2023 Section 18.5 Many Shuttles Allow Movement Across Mitochondrial Membranes • The respiratory chain regenerates NAD+ for use in glycolysis, but the inner mitochondrial membrane is impermeable to NADH and NAD+. • glycerol 3-phosphate shuttle = one means of transporting electrons from NADH into the electron transport chain • When cytoplasmic NADH transported by this shuttle is oxidized by the respiratory chain, 1.5 ATP are formed – because FAD rather than NAD+ is the electron acceptor Glycerol 3-Phosphate Shuttle Steps • Step 1: Electrons from NADH are transferred to dihydroxyacetone phosphate to form glycerol 3phosphate. • Step 2: Glycerol 3-phosphate is moved into the mitochondrion and reoxidized to dihydroxyacetone phosphate. – Electrons pass to an FAD prosthetic group, forming FADH2. • Step 3: Reduced flavin transfers its electrons to a molecule of Q, which enters the respiratory chain as QH2. The Glycerol 3-Phosphate Shuttle Allows for Rapid Rates of Oxidative Phosphorylation at the Cost of Energetic Efficiency The Malate–Aspartate Shuttle • the malate–aspartate shuttle = transports electrons from cytoplasmic NADH into mitochondria – forms mitochondrial NADH – in the heart and liver – mediated by two membrane carriers and four enzymes Malate–Aspartate Shuttle Steps • Step 1: Electrons are transferred from NADH in the cytoplasm to oxaloacetate, forming malate and NAD+. • Step 2: malate traverses the inner mitochondrial membrane in exchange for α-ketoglutarate. • Step 3: In the matrix, malate is then reoxidized by NAD+, forming oxaloacetate and NADH to form NADH • Step 4: Glutamate donates an amino group to oxaloacetate, forming aspartate and α-ketoglutarate. • Step 5: Aspartate and α-ketoglutarate enter the cytoplasm. • Step 6: In the cytoplasm, oxaloacetate is regenerated, and the cycle is restarted. The Malate–Aspartate Shuttle Preserves All the Chemical Potential Energy of Cytoplasmically Generated NADH The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP by ATPADP Translocase • ATP-ADP translocase (adenine nucleotide translocase, ANT) = specific transport protein that enables the exchange of cytoplasmic ADP for mitochondrial ATP – constitutes 15% of the protein of the inner mitochondrial membrane • ATP and ADP bind to ANT without Mg2+. • Inhibition of ANT leads to the inhibition of cellular respiration.     ADP3cytoplasm  ATP4matrix  ADP3matrix  ATP 4cytoplasm The ATP-ADP Translocase Catalyzes the Exchange of Entry of ADP and ATP • The translocase contains a single nucleotide-binding site that alternately faces the matrix and the cytoplasmic sides of the membrane. The Phosphate Carrier and the ATP Synthasome • phosphate carrier = mediates the exchange of cytoplasmic H2PO4− for mitochondrial OH− – works in concert with ANT to exchange cytoplasmic ADP and Pi for matrix ATP • ATP synthasome = a large complex composed of ATP synthase and the two transporters Multiple Structurally Homologous Mitochondrial Transporters Carry Specific Metabolites Across the Inner Mitochondrial Membrane What class of carrier protein do these mitochondrial transporters belong to? (1 of 2) © Macmillan Learning, 2023 What class of carrier protein do these mitochondrial transporters belong to? (2 of 2) antiporter © Macmillan Learning, 2023 Section 18.6 The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP • The ATP needs of the cell determine the rate of respiratory pathways and their components. • Approximately 30 molecules of ATP formed when glucose is completely oxidized to CO2. – 26 are formed in oxidative phosphorylation. – 2 are formed in the citric acid cycle. – 2 are formed in glycolysis. ATP Yield from the Complete Oxidation of Glucose TABLE 18.4 ATP yield from the complete oxidation of glucose Reaction sequence Glycolysis: Conversion of glucose into pyruvate (in the cytoplasm) Phosphorylation of glucose Phosphorylation of fructose 6-phosphate Dephosphorylation of 2 molecules of 1,3-BPG Dephosphorylation of 2 molecules of phosphoenolpyruvate 2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate Conversion of pyruvate into acetyl CoA (inside mitochondria) 2 molecules of NADH are formed Citric acid cycle (inside mitochondria) 2 molecules of adenosine triphosphate are formed from 2 molecules of succinyl CoA 6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, α-ketoglutarate, and malate 2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate ATP yield per glucose molecule –1 –1 +2 +2 +2 Oxidative phosphorylation (inside mitochondria) 2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the glycerol 3-phosphate shuttle) +3 2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP 2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP +5 +3 +15 +30 6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP Net Yield per Molecule of Glucose Information on the ATP yield of oxidative phosphorylation is from values given in P. C. Hinkle, M. A. Kumar, A. Resetar, and D. L. Harris, Biochemistry 30:3576,1991. Note: The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier value of 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport should be regarded as estimates. About 2 more molecules of ATP are formed per molecule of glucose oxidized when the malate–aspartate shuttle rather than the glycerol 3-phosphate shuttle is used. Which process yields the most ATP? (1 of 2) TABLE 18.4 ATP yield from the complete oxidation of glucose Reaction sequence Glycolysis: Conversion of glucose into pyruvate (in the cytoplasm) Phosphorylation of glucose Phosphorylation of fructose 6-phosphate Dephosphorylation of 2 molecules of 1,3-BPG Dephosphorylation of 2 molecules of phosphoenolpyruvate 2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate Conversion of pyruvate into acetyl CoA (inside mitochondria) 2 molecules of NADH are formed Citric acid cycle (inside mitochondria) 2 molecules of adenosine triphosphate are formed from 2 molecules of succinyl CoA 6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, α-ketoglutarate, and malate 2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate ATP yield per glucose molecule –1 –1 +2 +2 +2 Oxidative phosphorylation (inside mitochondria) 2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the glycerol 3-phosphate shuttle) +3 2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP 2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP +5 +3 +15 +30 6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP Net Yield per Molecule of Glucose Information on the ATP yield of oxidative phosphorylation is from values given in P. C. Hinkle, M. A. Kumar, A. Resetar, and D. L. Harris, Biochemistry 30:3576, 1991. Note: The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier value of 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport should be regarded as estimates. About 2 more molecules of ATP are formed per molecule of glucose oxidized when the malate–aspartate shuttle rather than the glycerol 3-phosphate shuttle is used. Which process yields the most ATP? (2 of 2) TABLE 18.4 ATP yield from the complete oxidation of glucose Reaction sequence Glycolysis: Conversion of glucose into pyruvate (in the cytoplasm) Phosphorylation of glucose Phosphorylation of fructose 6-phosphate Dephosphorylation of 2 molecules of 1,3-BPG Dephosphorylation of 2 molecules of phosphoenolpyruvate 2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate Conversion of pyruvate into acetyl CoA (inside mitochondria) 2 molecules of NADH are formed Citric acid cycle (inside mitochondria) 2 molecules of adenosine triphosphate are formed from 2 molecules of succinyl CoA 6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, α-ketoglutarate, and malate 2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate ATP yield per glucose molecule –1 –1 +2 +2 +2 Oxidative phosphorylation (inside mitochondria) 2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the glycerol 3-phosphate shuttle) +3 2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP 2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP +5 +3 +15 +30 6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP Net Yield per Molecule of Glucose Information on the ATP yield of oxidative phosphorylation is from values given in P. C. Hinkle, M. A. Kumar, A. Resetar, and D. L. Harris, Biochemistry 30:3576, 1991. Note: The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier value of 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport should be regarded as estimates. About 2 more molecules of ATP are formed per molecule of glucose oxidized when the malate–aspartate shuttle rather than the glycerol 3-phosphate shuttle is used. The Rate of Oxidative Phosphorylation Is Determined by the Need for ATP • Electrons do not flow through the electron-transport chain unless ADP is available to be converted into ATP. • The regulation of the rate of oxidative phosphorylation by ADP level is called respiratory (or acceptor) control. • At low ADP levels: – NADH and FADH2 are not consumed by the electrontransport chain. – the citric acid cycle slows because there is less NAD+ and FAD to feed the cycle. ATP Synthase Depends Upon the Rest of the ATP Synthesome, As Well As the ETC and CAC Oxidative Phosphorylation Can Be Inhibited at Many Stages • Many potent and lethal poisons inhibit oxidative phosphorylation in one of the following ways: – inhibition of the electron-transport chain, thus preventing generation of the proton-motive force – inhibition of ATP synthase – uncoupling electron transport from ATP synthesis – inhibition of ATP export 2-4-Dinitrophenol (DNP) and Xanthohumol • 2,4-dinitrophenol (DNP) = carries protons across the inner mitochondrial membrane, down their concentration gradient – ATP is not formed because the proton-motive force is continuously dissipated. • xanthohumol = promising drug that functions as a mild uncoupler and scavenges free radicals – may be used in the treatment of obesity and certain cancers Inhibitors of Electron Transport Block the Process at a Variety of Sites New Mitochondrial Diseases Are Constantly Being Discovered • Mitochondrial diseases are estimated to affect from 10 to 15 per 100,000 people. • Mitochondrial diseases are primarily inherited maternally. – human eggs harbor several hundred thousand molecules of mitochondrial DNA, whereas sperm only harbors a few hundred. • Mutations in Complex I are the most frequent cause of mitochondrial diseases. • Variations in the percentage of mitochondria with the mutation lead to large variations in the nature and severity of the symptoms, as well as the time of onset. Mitochondria Play a Key Role in Apoptosis • apoptosis = a form of programmed cell death – occurs in the course of development or in cases of significant cell damage – mitochondria act as control centers regulating the process • mitochondrial outer membrane permeabilization (MOMP) = process by which the outer membrane of damaged mitochondria becomes highly permeable – instigated by the Bcl family of proteins The Formation of the Apoptosome and the Activation of Caspases • cytochrome c = potent activator of apoptosis that interacts with apoptotic peptidase-activating factor 1 (APAF-1), leading to the formation of the apoptosome • apoptosome = protein complex that recruits and activates a proteolytic enzyme called caspase 9 • caspase 9 = member of the cysteine protease family that activates a cascade of other caspases – each caspase type destroys a particular target Power Transmission by Proton Gradients Is a Central Motif of Bioenergetics • Proton gradients power a variety of energy-requiring processes. • Proton gradients are a central, interconvertible currency of free energy in biological systems. Remember to complete your exit poll tonight! © Macmillan Learning, 2023