Lecture 28 –Quiz Friday on Beta-Oxidation of Fatty acids –Electron transport chain –Electron transport cofactors –ATPase

Page Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). 2.Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl- CoA 3.NAD + -dependent dehydrogenation of b- hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form  -ketoacyl- CoA. 4.C  -C  bond cleavage by  -ketoacyl-CoA thiolase (KT)

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 22-15aStructures of the common iron–sulfur clusters. (a) [Fe–S] cluster. Page 808

Figure 22-15bStructures of the common iron–sulfur clusters. (b) [2Fe–2S] cluster. Page 808

Figure 22-15cStructures of the common iron–sulfur clusters. (c) [4Fe–4S] cluster. Page 808

Figure 22-16X-Ray structure of ferredoxin from Peptococcus aerogenes. Page 809

Figure 22-17a Oxidation states of the coenzymes of complex I. (a) FMN. Page 810 Can accept or donate 1 or 2 e -

Figure 22-17b Oxidation states of the coenzymes of complex I. (b) CoQ. Page 810 CoenzymeCoenzyme Q’s hydrophobic tail allows it to be soluble in the inner membrane lipid bilayer.

Figure 22-21aVisible absorption spectra of cytochromes. (a) Absorption spectrum of reduced cytochrome c showing its characteristic , , and  (Soret) absorption bands. Page 813

Figure 22-21bThe three separate  bands in the spectrum of beef heart mitochondrial membranes indicate the presence of cytochromes a, b, and c. Page 813

Figure 22-22aPorphyrin rings in cytochromes. (a) Chemical structures. Page 813

Figure 22-22bPorphyrin rings in cytochromes. (b) Axial liganding of the heme groups contained in cytochromes a, b, and c are shown. Page 813

Complex I NADH-CoQ Oxidoreductase (NADH dehydrogenase) Electron transfer from NADH to CoQ More than 30 protein subunits - mass of 850 kD 1 st step is 2 e - transfer from NADH to FMN FMNH 2 converts 2 e - to 1 e - transfer 6-7 FeS clusters. Four H + transported out per 2 e- NADH + H + FMN Fe 2+ S CoQ NAD + FMNH 2 Fe 3+ SCoQH 2

Succinate FAD Fe 2+ S CoQ FumarateFADH 2 Fe 3+ SCoQH 2 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 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 UQH 2 and Complex III. UQ UQ.- UQH 2

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 UQH 2 ) CoQH 2 cyt b ox Fe 2+ S cyt c 1 ox cyt c red CoQcyt b red Fe 3+ S cyt c 1 red cyt c ox

cyt c red cyt a ox cyt a 3 red O2O2 cyt c ox cyt a red cyt a 3 ox 2 H 2 O Complex IV Cytochrome c Oxidase Electrons from cyt c are used in a four-electron reduction of O 2 to produce 2H 2 O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! Cytochrome c oxidase utilizes 2 hemes (a and a 3 ) 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 (Fe 3+ ) of a 3 Oligomycin and DCCD are ATP synthase inhibitors

Electron transport is coupled to oxidative phosphorylation

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= pH in – pH out ) 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  –2.303nRT  pH  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 = (0.059)(0.5V)  p = 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 = nF  P = (1)(96.48kJ/Vmole)(-0.2V) = -19 kJ/mole To make 1 ATP need 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 drives ATP synthesis! Two parts: F 1 and F 0

Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin

ADP + Pi ATP + H 2 O In catalytic site K eq = 1 ATP formation is easy step But once ATP is formed, it binds very tightly to catalytic site (binding constant = M) Proton induced conformation change weakens affinity of active site for ATP (binding constant = ) 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 120 o per ATP.

How does proton flow cause rotation?

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

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. Transport of ATP, ADP and Pi

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 (FADH 2 ), 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 107 o F temperatures