Oxidative Phosphorylation Glycogenolysis

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate pyruvate dehydrogenase (ox-decarbox; S-ester) Krebs’ Cycle How did he figure it out? Overview 8 Steps Energetics Regulation Summary Citrate Synthase (C–C) Aconitase (=, -OH) Isocitrate dehydrogenase (ox-decarbox; =O) Ketoglutarate dehydrogenase (ox-decarbox; S-ester) Succinyl-CoA synthetase (sub-level phos) Succinate dehydrogenase (=) Fumarase ( -OH) Malate dehydrogenase (=O) Oxidative Phosphorylation Energetics Mitochondria Transport Electron transport Discovery Four Complexes Oxidative Phosphorylation 1 Oxidative Phosphorylation Glycogenolysis Glycolysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative Oxidative Phosphorylation Learning goals: • Function of electron-transport chain in mitochondria…... make water (finish the reaction: C6H12O6 + 6O2 à 6CO2 + 6H2O) • Building up the proton-motive force • Synthesis of ATP in mitochondria, chloroplasts, & bacteria • Fuels for the cell, in the form of reduced carbon compounds (sugars), have been burned to carbon dioxide. • Electrons from reduced fuels are transferred to cofactors, which get reduced as NADH or FADH2. • In oxidative phosphorylation, energy from NADH and FADH2 is used to make ATP. …... But how? 2 Oxidative Phosphorylation Energy of the reduced cofactors Is there enough energy in NADH & FADH2 to drive the synthesis of ATP? Each ATP synthesis is about +7.3 kcal/mol (opposite of hydrolysis) We can do this calculation two ways: 1. Calculate the DE°’ needed to get 1 ATP made: compare to DE°’ of NADHàO2 2. Calculate the DG°’ for the NADHàO2 : compare to the DG°’ for ATP synthesis #1 ’ DE° of ATP DG°’ = –n F DE°’ DG°’ = DE ’ _____ ° –n F +7.3 kcalmol-1 __________________ = –0.16V –(2)(23.06V-1kcalmol-1) DE°’ of ATP = –0.16V DE°’ of NADHà ½ O2 DE°’ = E°’ (reduction) – E°’ (oxidation) = +0.82 V – (–0.32 V) = +1.14 V 7.1 times more energy in 2e– than needed to drive the synthesis of ATP Oxidative Phosphorylation Energy of the reduced cofactors DG°’ = –n F DE ’ ° Nernst Equation 3 Oxidative Phosphorylation Energy of the reduced cofactors Is there enough energy in NADH & FADH2 to drive the synthesis of ATP? Each ATP synthesis is about +7.3 kcal/mol (opposite of hydrolysis) We can do this calculation two ways: 1. Calculate the DE°’ needed to get 1 ATP made: compare to DE°’ of NADHàO2 2. Calculate the DG°’ for the NADHàO2 : compare to the DG°’ for ATP synthesis DG°’ of NADHà ½ O2 #2 DG°’ of ATP [NADH + H+ + ½ O2 ⇌ NAD+ + H2O] DE°’ = E°’ (reduction) – E°’ (oxidation) = +0.82 V – (–0.32 V) = +1.14 V 7.2 times more energy in 2e– going from NADH to oxygen than needed to drive the synthesis of ATP DG°’ = –n F DE ’ ° = –(2)(23.06V-1kcalmol-1)(+1.14V) DG°’ = –52.6 kcal/mol 6.5 times more energy in 2e– going from FADH2 to oxygen than needed to drive the synthesis of ATP DG°’ = +7.3 kcal/mol Oxidative Phosphorylation Structure of Mitochondria cow artery endothelial cell Double membrane leads to four distinct compartments: 1. Outer membrane: – relatively porous membrane; allows passage of metabolites 2. Intermembrane space (IMS): – – cow heart muscle 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. cow liver 4. Matrix – – location of the citric acid cycle and parts of lipid and amino-acid metabolism lower proton concentration D! (higher pH) ----- ≈ –0.06 V 4 Oxidative Phosphorylation • These translocases cost the membrane potential, which must be restored; costs 25% of Electron Transport. 14% of protein in the inner membrane is the ADP/ATP translocase • These translocases require energy from both the electrochemical gradient across inner mitochondrial membrane plus the proton gradient. Together they are called the Proton Motive Force. D! ≈ –0.06 V This antiporter works much like GLUT1. The import of ADP and export of ATP is favored by 1) the [ATP] concentrations, and 2) the charge difference. –1 0 • What generates this gradient? This symporter has to combat • the charge difference of Pi going to a more negative space, but its favored by the overwhelming high [H+] and [Pi] on the outside. How much energy does it take to pump H+ out? Oxidative Phosphorylation Energy required to pump a single proton against a pH gradient [H+out] [H+]out DG’ = RTln ____ + zF D" [H+]in ----Top [H+in] ln[H+]out = 2.3log[H+]out = -2.3 pHout Bottom ln(1/[H+]in) = 2.3-log[H+]in = +2.3 pHin pHout ≈ 6.75 pHin ≈ 7.5 And, if its more positive H+in ⇌ H+out +++++ D" ≈ –0.06 V D So, if its more negative = RT2.3(pHin – pHout) = 5.7(7.5 – 6.75) = 5.7(0.75) Switch the sign here because reaction is opposite that of transport = zF D" = (+1)(96480) D" = (+1)(96480)(+0.06) = 5.8 kJ/mol = 1.4 kcal/mol DG’ = 1.0 + 1.4 = 2.4 kcal/mol = 4.3 kJ/mol As a consequence, it will = 1.0 kcal/mol take ~3 protons per ATP. 5 Oxidative Phosphorylation P/O ratios Using tissues rich in mitochondria like pigeon muscle, and eventually using isolated mitochondria, biochemists would add different carbon compounds and measure two things: 1) Oxygen consumption 2) Amount of ATP made The ratio of ATP synthesized to the oxygen (½O2) consumed was termed the P/O ratio The ½O2 represents 2e– going through electron transport. The P/O ratio for various fuel molecules provided to cells/mitochondria were measures as: DG°’ of NADHà ½ O2 DG°’ = –52.6 kcal/mol DG°’ = +7.3 kcal/mol NADH was ~3 Pyruvate was ~3 Succinate was ~2 Ascorbate was 1 22/53 = 42% efficient 7.3 x P/O of 3 = 22 kcal/mol This is the energy recovered from the complete oxidation What makes this proton motive force? Electron Transport 6 Electron Transport Electron-Transport Chain Complexes Contain a Series of Electron Carriers • When it was realized that isolated mitochondria are capable of respiration (oxygen consumption when provided fuels), biochemists began purifying them and their components. • The first things purified were redox compounds and small stable proteins: – – – – – – NADH flavin mononucleotide (FMN) flavin adenine dinucleotide (FAD)(bound to protein; flavoproteins) iron-sulfur clusters Coenzyme Q (Ubiquinol) cytochromes a, b, or c • Once purified, they were analyzed by meaBig Drop! suring their E ’. ° Big Drop! • Order of transfer of electrons is dependent Big Drop! on E ’ : ° E°’ FAD-E + 2 H+ + 2e– à FADH2 -E –0.02 FeS Electron Transport • NAD+, FMN, and FAD accept electrons. The flavin nucleotides can accept one or two electrons, and can also donate one electron at a time to acceptors that can only accept single electrons • Ubiquinone, also called Coenzyme Q, is an isoprene lipid that readily accepts electrons. Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol (CoQH2). Its found IN the inner membrane. • Iron-sulfur complexes (Fe-S) that can only carry one electron at a time (role is different than in aconitase). 7 Cytochromes Electron Transport • Small one-electron carrier proteins Cytochromes • Iron-coordinating porphyrin-ring derivatives • b/b1, c, or a/a3 differ by ring additions Electron Transport Cytochrome c • Mobile electron carrier; a peripheral membrane protein – Cytochrome c moves through the intermembrane space (IMS). • A soluble heme-containing protein • Heme iron can be either ferrous (Fe2+, reduced) or ferric (Fe3+, oxidized). • Cytochrome c carries a single electron. • The two redox forms have different spectra: • Intense Soret band near 400 nm absorbs blue light and gives cytochrome c an intense red color. 8 Electron Transport à O2 Big Drop! Inhibitors of Electron Transport Big Drop! • Inhibitors all stop ET and ATP synthesis: very toxic! Spectral work Cyto-a3 Cyto-b Cyto-c Big Drop! NADH Cyto-c1 Cyto-a Flavin Fully reduced Cyto-c + rotenone + antimycin A + CN– 300 350 400 450 500 550 600 650 700 Electron Transport Electron-Transport Chain Complexes Contain a Series of Electron Carriers rotenone malonate Antimyciin A cyanide • Better techniques for isolating and handling mitochondria, and isolated various fractions of the inner mitochondrial membrane • Measure E°’ • They corresponded to these large drops, and they contained the redox compounds isolated previously. • When assayed for what reactions they could perform, they could perform certain redox reactions and not others. • When isolated, including isolating the individual redox compounds, and measuring the E°’ for each, it was clear that an electron chain was occurring; like a wire! • Lastly, when certain inhibitors were added, some of the redox reactions could be inhibited and others not. Site of the inhibition could be mapped. 9