Oxidative Phosphorylation - Chapter 19 PDF

Oxidative phosphorylation Marc A. Ilies, Ph. D. Lehninger - Chapter 19 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAI...

Oxidative phosphorylation Marc A. Ilies, Ph. D. Lehninger - Chapter 19 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAIlies 2024 1  Energy from reduced fuels is used to synthesize ATP via cellular respiration Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell. Electrons from reduced fuels are transferred to reduced cofactors NADH or FADH2. In oxidative phosphorylation, energy from NADH and FADH2 is used to make ATP.  Chemiosmotic Theory  ADP + Pi  ATP is highly thermodynamically unfavorable.  Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient. G is directly related to ΔE = Eo (e- acceptor) – Eo (e- donor)  The energy released by electron transport (redox processes) is used to transport protons against the electrochemical gradient (chemiosmotic theory, Peter Mitchell, 1961)  Chemiosmotic energy coupling requires membranes The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions. – plasma membrane in bacteria – inner membrane in mitochondria – thylakoid membrane in chloroplasts The membrane must contain proteins that couple the “downhill” flow of electrons in the electron-transfer chain with the “uphill” flow of protons across the membrane. The membrane must also contain a protein that couples the “downhill” flow of protons to the phosphorylation of ADP.  Structure of a Mitochondrion Double membrane leads to four distinct compartments: 1. Outer membrane: – relatively porous membrane; allows passage of metabolites 2. Intermembrane space (IMS): – similar environment to cytosol – higher proton concentration (lower pH) 3. Inner membrane – relatively impermeable, with proton gradient across it – location of electron transport chain complexes – convolutions called cristae serve to increase the surface area. 4. Matrix – location of the citric acid cycle and parts of lipid and amino acid metabolism – lower proton concentration (higher pH)  Electron-Transport Chain Complexes Contain a Series of Electron Carriers Each complex contains multiple redox centers consisting of: – flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) – cytochromes a, b, or c – iron-sulfur clusters Order of transfer of electrons is dependent on reduction potential: Tendency to accept e- increases  NAD(P)+/NAD(P)H, FMN/FMNH2 FAD/FADH2 electron carriers Coenzymes associated with dehydrogenases - transfers hydride ions: H- (one proton plus two electrons H+ + 2.e-) - deficiency: Pellagra - transfers hydrogen atoms.H (one proton plus one electron H+ +.e-) - deficiency: Ariboflavinosis  Coenzyme Q or Ubiquinone Ubiquinone is a lipid-soluble quinone isoprenoid compound that readily accepts electrons from different redox- active compounds Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol. Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side. Coenzyme Q is a mobile electron carrier transporting electrons from Complexes I and II to Complex III. O OH + 2 e - + 2 H+ O OH 1,4-benzoquinone hydroquinone  Cytochromes One-electron carriers based on Fe3+/Fe2+ redox system Fe3+/Fe2+ coordinating porphyrin ring (heme) derivatives a, b, or c differ by ring additions; substitution modulates redox properties:  Iron-Sulfur Proteins One-electron carriers based on Fe3+/Fe2+ redox system Iron ions coordinated by cysteines in the protein or assembled in iron-sulfur clusters that are coordinated by Cys residues in the protein Iron-sulfur clusters contain an equal number of iron and sulfur atoms  Chemiosmotic Model for ATP Synthesis Electron transport through complexes I→IV sets up a proton-motive force (flow of electrons creates a concentration gradient of protons) Energy of proton-motive force drives synthesis of ATP. TABLE 19-3 The Protein Components of the Mitochondrial Respiratory Chain Enzyme complex/protein Mass (kDa) Number of subunitsa Prosthetic group(s) I NADH dehydrogenase 850 45 (14) FMN, Fe-S II Succinate dehydrogenase 140 4 FAD, Fe-S III Ubiquinone: cytochrome c oxidoreductaseb 250 11 Hemes, Fe-S Cytochrome cc 13 1 Heme IV Cytochrome oxidaseb 204 13 (3–4) Hemes; CuA, CuB a Number of subunits in the bacterial complexes in parentheses. b Mass and subunit data are for the monomeric form. c Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein.  Flow of electrons in mitochondria Transport of a pair of electrons from NADH to O 2 is achieved through successive redox reactions, in Complexes I to IV The energetic difference in redox potential is gradually harvested to create a proton concentration gradient Transport of a pair of electrons from NADH to O2 produces 220 kJ; It takes ~ 30kJ to produce one ATP. This is more than enough energy to make 3 ATP. The rest of energy is lost as heat The sequence can be determined using inhibitors of electron transfer Uncouplers can bypass the H+ flow through ATPase  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 a time toward the ubiquinone binding site. Transfer of two electrons from NADH to ubiquinone is accompanied by a transfer of protons from the matrix (N) to the intermembrane space (P); about four protons are transported per one NADH. NADH + Q + 5H+N = NAD+ + QH  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, which becomes reduced QH2. Does not transport protons FADH2 + Q FAD + QH2 Succinate dehydrogenase is a single enzyme with dual role: convert succinate to fumarate in the citric acid cycle capture and donate electrons in the electron transport chain  Ubiquinone:Cytochrome c Oxidoreductase, a.k.a. Complex III Uses two electrons from QH2 to reduce two molecules of cytochrome c Additionally contains iron- sulfur clusters, cytochrome b, and cytochrome c Clearance of electrons from the reduced quinones via the Q- cycle results in translocation of four additional protons to the intermembrane space:  Cytochrome c The second mobile electron carrier – Ubiquinone moves through the membrane. – Cytochrome c moves through the intermembrane space. 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. absorbs blue light and gives cytochrome c an intense red color; named by the position of their longest-wavelength (α) peak.  Cytochrome Oxidase, a.k.a. Complex IV Mammalian cytochrome oxidase is a membrane protein with 13 subunits. Contains two heme groups: a and a3 Contains copper ions – CuA: two ions that accept electrons from cyt c – CuB: bonded to heme a3, forming a binuclear center that transfers four electrons to oxygen Four electrons are used to reduce one oxygen molecule into two water molecules. Four protons are picked up from the matrix in this process. Four additional protons are passed from the matrix to the intermembrane space: 4 cyt c (reduced) + 8H+N + O2 → 4 cyt c (oxidized) + 4H+P + 2 H2O Summary of the Electron Flow in the Respiratory Chain Complex I  Complex III  Complex IV NADH + 11H+(N) + ½O2 ——> NAD+ + 10H+(P) + H2O Complex II  Complex III  Complex IV FADH2 + 6H+(N) + ½O2 ——> FAD + 6H+(P) + H2O Difference in H+ transported will reflect difference in ATP synthesized (3 for NADH, 2 for FADH2)  Mitochondria can generate reactive oxygen species ROS - when the rate of electron entry into the respiratory chain and the rate of electron transfer through the chain are mismatched, superoxide radical ( O2-) and ROS production increases via e- transfer from ubiquinone: (SOD) - cell can correct free-radical production via superoxide dismutase (SOD) and glutathione peroxidase, with the use of Glutathione (GSH) - RBCs do not have mitochondria!  Proton-Motive Force The proteins in the electron- transport chain created the electrochemical proton gradient by one of three means: – actively transporting protons across the membrane Complex I and Complex IV – chemically removing protons from the matrix reduction of Q and reduction of oxygen – releasing protons into the intermembrane space oxidation of QH2  Proton-motive force drives ATP synthesis - the electrochemical energy stored in the H+ concentration gradient and the separation of charge across the inner mitochondrial membrane (the proton motive force) drives ATP synthesis via ATP synthase:  Mitochondrial ATP Synthase Complex Contains two functional units: F 0 integral membrane complex transports protons from IMS to matrix, dissipating the proton gradient energy transferred to F to catalyze 1 phosphorylation of ADP F1 soluble complex in the matrix individually catalyzes the hydrolysis of ATP Structure: Hexamer arranged in three αβ dimers Dimers can exist in three different conformations: open: empty loose: binding ADP and Pi tight: catalyzes ATP formation and binds product  Synthesis of ATP in ATP synthase Translocation of three protons fuels synthesis of one ATP: https://www.youtube.com/watch?v=b_cp8MsnZFA Chemiosmotic Model for ATP Synthesis  Electron transport and ATP synthesis are coupled As described, ATP synthesis requires electron transport However, electron transport also requires ATP synthesis - Addition of both ADP + P and succinate is needed for mitochondrial respiration; ATP is synthesized. - Addition of cyanide (CN-), which blocks electron transfer between cytochrome oxidase (Complex IV) and O2, inhibits both respiration and ATP synthesis. - Vice-versa, mitochondria provided with succinate respire and synthesize ATP only when ADP and Pi are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. - Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis.  Uncouplers Transport of protons across mitochondrial membrane by 2,4-dinitrophenol (DNP): Valinomycin is a K+ Ionophore - Ionophores will disrupt ion gradients across membranes and thus uncouple electron transport. - Experimentally an artificially imposed electrochemical gradient can be produced by the addition of a + charge from K+, this can drive ATP synthesis in the absence of an oxidizable substrate as electron donor - This provides evidence of the role of a proton gradient in ATP synthesis. Valinomycin 26  Uncouplers Thermogenin in Brown Fat - Uncoupling protein in the mitochondria of brown adipose tissue - Provides heat via H+ flow through it - Provides heat for babies (have high surface area/volume ratio), also in hibernating animals 27  * * * * * * * ** 28  Transport of different species in/out of matrix - Translocation of a fourth proton per ATP is required to facilitate cotransport of substrates into and products out of the mitochondria:  Malate-Aspartate Shuttle - this shuttle transports reducing equivalents (NADH) from cytosol into the mitochondrial matrix (liver, kidneys, heart):  Glycerol-3-Phosphate Shuttle - This alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in skeletal muscle and the brain: - Note that from NADH we get FADH2, so we are losing energy (1 ATP) – see next slide!  Glucose 2H++ 2e- 1/2 O 2 Pyruvate ADP 2H++ 2e- ADP ADP 2H++ 2e- NADH FMN CoQ b c1 c a isocitrate Acetyl CoA ATP ATP ATP -ketoglutarate citrate 2H++ 2e- oxaloacetate succinyl CoA malate succinate 2H++ 2e- FADH2 fumarate 2H++ 2e- Glycolysis 2 substrate level phosphoryations-2ATP = 2 ATP Oxidation of 2 NADH from glycolysis 2 x 3(Malate Shuttle) = 6 ATP Pyruvate acetyl-CoA 2 xNADH = 6 ATP 6 x NADH = 18 ATP Krebs 2 x FADH 2 = 4 ATP 2 substrate level phosphorylations = 2 ATP 38 ATP produced using Malate Shuttle !! Total = 38 ATP 36 ATP produced using Glycerol PO4 Shuttle !! 27  Regulation of Oxidative Phosphorylation Primarily regulated by substrate availability – NADH and ADP/Pi The rate of O2 consumption and thus ATP synthesis is regulated by the amount of ADP ( i.e. the Pi acceptor). The intracellular concentration of ADP is a measure of the energy status of the cell. Another measure is the Mass-Action Ratio: [ATP] / ([ADP] [Pi]) Normally this ratio is high. When energy is required the ratio falls so [ADP] increases, respiration increases and more ATP is produced. The ratio is fairly stable so that ATP is formed only as fast as needed. High ATP has inhibitory effects at multiple levels Inhibition of OxPhos leads to accumulation of NADH. – causes feedback inhibition cascade up to PFK-1 in glycolysis  Mitochondria can play an initiating role in apoptosis - loss of mitochondrial membrane integrity releases cytochrome c in the cytosol, where it acts as a trigger for apoptosis (programmed cell death) by stimulating the activation of a family of proteases called caspases  Mitochondria genetics is very important Mitochondrial DNA is circular and well conserved. The mitochondrial genome has 37 genes and encodes rRNA, tRNA, and enzymes in central metabolism, including the citric acid cycle and oxidative phosphorylation. – Mitochondria have their own ribosomes to allow synthesis of proteins within the organelle. – However, the vast majority of mitochondrial proteins are encoded on nuclear DNA, synthesized in the cytosol, and imported into the mitochondria. Mitochondrial DNA is maternally inherited. Mutations can cause many diseases: Mitochondrial Mutations Result in a Rare form of Diabetes Defects in oxidative phosphorylation result in low [ATP] in the cell. – Insulin cannot be released from the cell.  Goals and Objectives Upon completion of this lecture at minimum you should be able to answer the following: ►What is the role of cellular respiration in catabolism and ATP synthesis? ►What are the implications of the chemiosmotic theory in terms of energetics, mitochondria design? What processes take place in mitochondria and where? ►Which are the main redox intermediates (electron carriers) in oxdative phosphorylation, what are their details and what role they play in oxPhos? ►What complexes involve the electron flow in mitochondrial, what is their role and what are their particularities? ►How the proton motive force is created (sources) and how it is used to produce ATP? What enzymes are involved, what is their role and particularities? ►Which are the inhibitors of electron flow, of ATP synthesis, at what steps they act, how is the electron flow coupled with ATP production, how can it be uncoupled ? What particularities have different uncouplers? ►What are the particularities of material (compounds, intermediates) transfer in/out of mitochondria? ►How is oxidative phosphorylation regulated? ►What is the role of mitochondria in ROS production and apoptosis, what are the details of mitochondria genetics and what diseases can be observed as a result of mutations of different mitochondrial enzymes? 37