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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
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



Cofactors of the electron transport chain electron carriers.

  • Fe-S clusters

  • Coenzyme Q (ubiquinone)

  • Flavin mononucleotide

  • FAD

  • Cytochrome a

  • Cytochrome b

  • Cytochrome c

  • CuA

  • CuB


Iron-sulfur clusters electron carriers.

  • 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
Figure 22-15a electron carriers. 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
Figure 22-15b electron carriers. 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
Figure 22-15c electron carriers. Structures of the common iron–sulfur clusters. (c) [4Fe–4S] cluster.

Page 808


Figure 22 16 x ray structure of ferredoxin from peptococcus aerogenes
Figure 22-16 electron carriers. X-Ray structure of ferredoxin from Peptococcus aerogenes.

Page 809


Figure 22 17a oxidation states of the coenzymes of complex i a fmn
Figure 22-17a electron carriers. 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
Figure 22-17b electron carriers. 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 electron carriers. 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 electron carriers. 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
Figure 22-22a electron carriers. Porphyrin rings in cytochromes. (a) Chemical structures.

Page 813


Figure 22-22b electron carriers. Porphyrin rings in cytochromes. (b) Axial liganding of the heme groups contained in cytochromes a, b, and c are shown.

Page 813


NADH + H electron carriers.+

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 electron carriers.

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
Q-Cycle electron carriers.

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


CoQH electron carriers.2

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 electron carriers.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 electron carriers.

  • 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 electron carriers.

  • 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
Proton Motive Force ( electron carriers.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 –2.303nRTpH

  • p = p = G/nF

  • p =  –(0.059) pH


Proton motive force p1
Proton Motive Force ( electron carriers.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 p2
Proton Motive Force ( electron carriers.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 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 electron carriers.

  • 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
Binding Change Mechanism vesicles containing the ATP synthase and bacteriorhodopsin

  • 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 mechanism1
Binding Change Mechanism vesicles containing the ATP synthase and bacteriorhodopsin

  • 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
ATPase is a Rotating Motor vesicles containing the ATP synthase and bacteriorhodopsin

  • 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.


How does proton flow cause rotation
How does proton flow cause rotation? vesicles containing the ATP synthase and bacteriorhodopsin


Active transport of atp adp and pi across mitochondrial inner membrane
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
Transport of ATP, ADP and Pi Inner Membrane

  • 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 Inner Membrane

  • 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
P/O Ratio Inner Membrane

  • 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
Regulation of Oxidative Phosphorylation Inner Membrane

  • 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 Inner Membrane

  • 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


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