Lecture 28

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

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1. 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 22-15a Structures of the common iron–sulfur clusters. (a) [Fe–S]

cluster.

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

cluster.

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

cluster.

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

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Figure 22-17aOxidation states of the

coenzymes of complex I. (a) FMN.

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Can accept or donate 1 or 2 e-

Figure 22-17bOxidation states of the coenzymes of

complex I. (b) CoQ.

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Coenzyme Q’s hydrophobic tail allows it to be soluble in the inner membrane lipid bilayer.

Figure 22-21a Visible absorption spectra of cytochromes. (a) Absorption spectrum of reduced cytochrome c showing its

characteristic , , and (Soret) absorption bands.

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Figure 22-21b The three separate bands in the spectrum of beef heart mitochondrial membranes indicate the

presence of cytochromes a, b, and c.

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Figure 22-22a Porphyrin rings in cytochromes. (a) Chemical structures.

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

are shown.

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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-

NADH + H+

         FMN

         Fe2+S

         CoQ

NAD+ FMNH2 Fe3+S CoQH2

Succinate          

FAD          

Fe2+S          

CoQ

Fumarate FADH2 Fe3+S CoQH2

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 UQH2 and Complex III.

UQ

UQ.-

UQH2

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)

                             CoQH2 cyt b ox Fe2+S cyt c1 ox cyt c red

CoQ cyt b red Fe3+S cyt c1 red cyt c ox

cyt c red          

cyt a ox          

cyt a3 red          

O2

cyt c ox cyt a red cyt a3 ox 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

Electron transport is coupled to oxidative phosphorylation

Chemiosmotic TheoryObservations 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 drives ATP synthesis!

• Two parts: F1 and F0

Racker & Stoeckenius confirmed Mitchell’s hypothesis using 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 Mechanism

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.

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 (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

O2N OH

NO2 H

O2N O

NO2