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Lecture 28. Quiz Friday on Beta-Oxidation of Fatty acids Electron transport chain Electron transport cofactors ATPase. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).
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Lecture 28 • Quiz Friday on Beta-Oxidation of Fatty acids • Electron transport chain • Electron transport cofactors • ATPase
Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917
Standard reduction potentials of the major respiratory electron carriers.
Cofactors of the electron transport chain • Fe-S clusters • Coenzyme Q (ubiquinone) • Flavin mononucleotide • FAD • Cytochrome a • Cytochrome b • Cytochrome c • CuA • CuB
Iron-sulfur clusters • 4 main types of iron sulfur clusters • [2Fe-2S] and [4Fe-4S] cluster coordinated by 4 Cys SH • [3Fe-4S] is a [4Fe-4S] lacking one Fe atom. • [Fe-S] is only found in bacteria, liganded to 4 Cys • Rieske iron-sulfur proteins [2Fe-2S] cluster but 1 Fe is coordinated by 2 His. • Oxidized and reduced states of all Fe-S clusters differ by one formal charge.
Figure 22-15a Structures of the common iron–sulfur clusters. (a) [Fe–S] cluster. Page 808
Figure 22-15b Structures of the common iron–sulfur clusters. (b) [2Fe–2S] cluster. Page 808
Figure 22-15c Structures of the common iron–sulfur clusters. (c) [4Fe–4S] cluster. Page 808
Figure 22-16 X-Ray structure of ferredoxin from Peptococcus aerogenes. Page 809
Figure 22-17a Oxidation states of the coenzymes of complex I. (a) FMN. Can accept or donate 1 or 2 e- Page 810
Figure 22-17b Oxidation states of the coenzymes of complex I. (b) CoQ. Coenzyme Q’s hydrophobic tail allows it to be soluble in the inner membrane lipid bilayer. Page 810
Figure 22-21a Visible absorption spectra of cytochromes. (a) Absorption spectrum of reduced cytochrome c showing its characteristic a, b, and g (Soret) absorption bands. Page 813
Figure 22-21b The three separate bands in the spectrum of beef heart mitochondrial membranes indicate the presence of cytochromes a, b, and c. Page 813
Figure 22-22a Porphyrin rings in cytochromes. (a) Chemical structures. Page 813
Figure 22-22b Porphyrin rings in cytochromes. (b) Axial liganding of the heme groups contained in cytochromes a, b, and c are shown. Page 813
NADH + H+ CoQ NAD+ CoQH2 Complex I • NADH-CoQ Oxidoreductase (NADH dehydrogenase) • Electron transfer from NADH to CoQ • More than 30 protein subunits - mass of 850 kD • 1st step is 2 e- transfer from NADH to FMN • FMNH2 converts 2 e- to 1 e- transfer • 6-7 FeS clusters. • Four H+ transported out per 2 e- FMN Fe2+S FMNH2 Fe3+S
Succinate CoQ Fumarate CoQH2 FAD Fe2+S FADH2 Fe3+S Complex II • Succinate-CoQ Reductase • Contains the succinate dehydrogenase (from TCA cycle!) • four subunits • Two largest subunits contain 2 Fe-S proteins • Other subunits involved in binding succinate dehydrogenase to membrane and passing e- to Ubiquinone • FAD accepts 2 e- and then passes 1 e- at a time to Fe-S protein • No protons pumped from this step
Q-Cycle UQ • Transfer from the 2 e- carrier ubiquinone (QH2) to Complex III must occur 1 e- at a time. • Works by two single electron transfer steps taking advantage of the stable semiquinone intermediate • Also allows for the pumping of 4 protons out of mitochondria at Complex III • Myxothiazol (antifungal agent) inhibits electron transfer from UQH2 and Complex III. UQ.- UQH2
CoQH2 cyt c red CoQ cyt c ox cyt c1ox cyt b ox Fe2+S cyt c1red cyt b red Fe3+S Complex III • CoQ-Cytochrome c oxidoreductase • CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle • Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents • cyt c is a water-soluble electron carrier • 4 protons pumped out of mitochondria (2 from UQH2)
cyt c red cyt a ox cyt a3red O2 cyt c ox cyt a red cyt a3ox 2 H2O Complex IV • Cytochrome c Oxidase • Electrons from cyt c are used in a four-electron reduction of O2 to produce 2H2O • Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! • Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites • Complex IV also transports H+ (2 protons)
Inhibitors of Oxidative Phosphorylation • Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! • Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 • Oligomycin and DCCD are ATP synthase inhibitors
Chemiosmotic Theory • Observations to explain the chemiosmotic hypothesis • Oxidative phosphorylation requires intact inner mitochondrial membrane • The inner membrane is impermeable to charged ions (free diffusion would discharge the gradient) • Compounds that increase the permeabililty of the inner mitochondrial membrane to protons uncouple electron transport from oxidative phosphorylation.
Proton Motive Force (p) • PMF is the energy of the proton concentration gradient • The chemical (pH= pHin – pHout) potential and the electrical potential(= in – out) contribute to PMF • G = nf and G = –2.303nRT pH • G for transporting 1 H+ from inner membrane space to matrix = G = nf –2.303nRTpH • p = p = G/nF • p = –(0.059) pH
Proton Motive Force (p) • What contributes more to PMF, or pH? • In liver =-0.17V and pH=0.5 • p = –(0.059)pH = -0.17-(0.059)(0.5V) • p = -0.20 V • p=(-0.17V/-0.20V) X 100% = 85% • 85% of the free energy is derived form
Proton Motive Force (p) • How much free energy generated from one proton? • G = nFP = (1)(96.48kJ/Vmole)(-0.2V) = -19 kJ/mole • To make 1 ATP need 40-50 kJ/mole. • Need to translocate more than one proton to make one ATP (about 3 H+/ATP) • ETC translocates 10 protons per NADH
ATP Synthase • Proton diffusion through the protein drivesATP synthesis! • Two parts: F1 and F0
Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin
Binding Change Mechanism • ADP + Pi <-> ATP + H2O • In catalytic site Keq = 1 • ATP formation is easy step • But once ATP is formed, it binds very tightly to catalytic site (binding constant = 10-12M) • Proton induced conformation change weakens affinity of active site for ATP (binding constant = 10-5)
Binding Change Mechanism • Different conformation at 3 catalytic sites • Conformation changes due to proton influx • ADP + Pi bind to L (loose) site • Proton (energy) driven conformational change (loose site) causes substrates to bind more tightly (T). • ATP is formed in tight-site. • ATP is released from the O (open) site. • Requires influx of three protons to get one ATP
ATPase is a Rotating Motor • Bound subunits to glass slide • Attached a fluroescent actin chain to subunit. • Hydrolysis of ATP to ADP + Pi cause filament to rotate 120o per ATP.
Active Transport of ATP, ADP and Pi Across Mitochondrial Inner Membrane • ATP is synthesized in the matrix • Need to export for use in other cell compartments • ADP and Pi must be imported into the matrix from the cytosol so more ATP can be made. • Require the use of transporters
Transport of ATP, ADP and Pi • Adenine nucleotide translocator = ADP/ATP antiport. • Exchange of ATP for ADP causes a change in due to net export of –1 charge • Some of the energy generated from the proton gradient (PMF) is used here • Pi is imported into the matrix with a proton using a symport. • Because negative charge on the phosphate is canceled by positive charge on proton no effect on , but effects pH and therefore PMF.
Transport of ATP, ADP and Pi • NRG required to export 1 ATP and import 1 ADP and 1 Pi = NRG generated from influx of one proton. • Influx of three protons required by ATPase to form 1 ATP molecule. • Need the influx of a total of 4 protons for each ATP made.
P/O Ratio • The ratio of ATPs formed per oxygens reduced • e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen • 4 H+ flow back into matrix per ATP to cytosol • 10/4 = 2.5 for electrons entering as NADH • For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen • 6/4 = 1.5 for electrons entering as succinate
Regulation of Oxidative Phosphorylation • ADP is required for respiration (oxygen consumption through ETC) to occur. • At low ADP levels oxidative phosphorylation low. • ADP levels reflect rate of ATP consumption and energy state of the cell. • Intramolecular ATP/ADP ratios also impt. • At high ATP/ADP, ATP acts as an allosteric inhibitor for Complex IV (cytochrome oxidase) • Inhibition is reversed by increasing ADP levels.
Uncouplers • Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient • Uncouplers are hydrophobic molecules with a dissociable proton • They shuttle back and forth across the membrane, carrying protons to dissipate the gradient • w/o oxidative-phosphorylation energy lost as heat • Dinitrophenol once used as diet drug, people ran 107oF temperatures