20. electron transport and oxidative phosphorylation -...

20. Electron Transport and Oxidative Phosphorylation

20.1 What Role Does Electron Transport Play in Metabolism ?

◈ Electron transport

- Role of oxygen in metabolism as final acceptor of electrons

- In inner mitochondrial membrane

◈ Oxidative phosphorylation

- Production of ATP by oxidation of nutrients

- In mitochondria

◈ Definition

Fe(Ⅲ) + e- Fe(Ⅱ) (reduction)

Fe(Ⅱ) Fe(Ⅲ) + e- (oxidation)

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◈ Reactions of electron transport chain

- Coupled to synthesis of ATP

① Pumping of protons across inner mitochondrial membrane

② Creating a pH gradient

③ Storing potential energy


◈ Oxygen, ultimate electron acceptor, is reduced to water

- Complete series of oxidation-reduction reactions

20.2 What Are the Reduction Potentials for the Electron Transport Chain ?

◈ Energy changes in electron transport

- Movement of electrons from one carrier to another

- Reduction potential: whether electrons would be more likely to be transferred from NADH to coenzyme Q or the other way around ?

- Hydrogen ion is in aqueous solution in equilibrium with hydrogen gas.

2H+ + 2e- H2


- Hydrogen/H+ pair has a higher reduction potential than ethanol/ acetaldehyde pair.


- Electron flow in opposite direction (H2/H+ pair has a lower reduction potential.)

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◈ Example

- Reduction of oxygen : 0.816 volts

- If this reaction were paired directly with NAD+/NADH, what would happen ?

NADH + H+ NAD+ + 2H+ + 2e- 0.320

1/2O2 + 2H+ + 2e- H2O 0.816

Sum NADH + 1/2O2 + H+ NAD+ + H2O 1.136

- ΔG° of redox reaction

ΔG° = -nFΔE°’

ΔG° = -(2)(96.485 kJ V-1 mol-1)(1.136V) = -219 kJ mol-1

Very large number if NADH reduced oxygen directly

20.3 How Are the Electron Transport Complexes Organized ?

◈ Respiratory complexes

- Four mutienzyme systems

- Can be isolated from inner mitochondrial membrane

- Integral parts of inner mitochondrial membrane

Complex Ⅰ

◈ NADH-CoQ oxidoreductase

- Transfer of electrons from NADH to coenzyme Q (CoQ)

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Complex Ⅰ

- Flavoprotein has a flavin coenzyme or FMN (flavin mononucleotide).


Complex Ⅰ

◈ Reaction in several steps

- Successive oxidation and reduction of flavoprotein and iron-sulfur moiety

① NADH + H+ + E-FMN NAD+ + E-FMNH2

② E-FMNH2 + 2Fe-Soxidized E-FMN + 2Fe-Sreduced + 2H+

③ 2Fe-Sreduced + CoQ + 2H+ 2Fe-Soxidized + CoQH2

- Coenzyme Q as ubiquinone


Complex Ⅰ

- Overall equation

NADH + H+ + CoQ NAD+ + CoQH2

- Proton pumping create pH (proton) gradient


Complex Ⅰ

- Phosphorylation of ADP to ATP : ΔG°’ = -28.0 ~ -33.5 kJ mol-1.


Complex Ⅰ

◈ Some of carriers

- Carry electrons and hydrogens in reduced forms: NADH

- Carry only electrons: iron-sulfur protein


Complex Ⅱ

◈ Succinate-CoQ oxidoreductase

- Transfer of electrons to coenzyme Q

- Substrate is succinate from citric acid cycle.

Succinate + E-FAD Fumarate + E-FADH2

E-FADH2 + Fe-Soxidized E-FAD + Fe-Sreduced

Fe-Sreduced + CoQ + 2H+ Fe-Soxidized + CoQH2

- Overall reaction is

Succinate + CoQ Fumarate + CoQH2

- ΔG°’ = -13.5 kJ mol-1

- No ATP production, and no hydrogen ions are pumped out.


Complex Ⅲ

◈ Cytochromes

- Electrons are passed from CoQ to first of a series of very similar proteins.

- Successive oxidation-reduction reactions of cytochromes,

Fe(Ⅲ) + e- Fe(Ⅱ) (reduction)

Fe(Ⅱ) Fe(Ⅲ) + e- (oxidation)

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Complex Ⅲ

◈ CoQH2-cytochrome c oxidoreductase

- Oxidation of reduced coenzyme Q (CoQH2).

- Electrons are passed along to cytochrome c in a multistep process.

- Overall reaction is

CoQH2 + 2Cyt c[Fe(Ⅲ)] CoQ + 2Cyt c[Fe(Ⅱ)] + 2H+


Complex Ⅲ

- CoQ can exist in three forms.


Complex Ⅲ

◈ Q cycle

- One electron is passed from reduced coenzyme Q to iron-sulfur clusters to cytochrome c1.

CoQH2 Fe-S Cyt c1

- By omitting iron-sulfur proteins,

CoQH2 + Cyt c1(oxidized)

Cyt c1(reduced) + CoQ- (semiquinone anion) + 2H+

- Proton pumping and ATP production is coupled.

- ΔG°’ = -34.2 kj mol-1 for each mole of NADH.


Complex Ⅳ

◈ Cytochrome c oxidase

- Transfer of electrons from cytochrome c to oxygen

- Overall reaction is,

2Cyt c[Fe(Ⅱ)] + 2H+ + 1/2O2 2Cyt c[Fe(Ⅲ)] + H2O

- Proton pumping as a result

- Contain cytochrome a and a3, as well as two Cu2+ ions

Cyt c Cyt a Cu 2+ Cyt a3 O2


Complex Ⅳ

- Reactions of cytochromes more explicitly,

Cyt c [reduced, Fe(Ⅱ)] + Cyt aa3 [oxidized, Fe(Ⅲ)]

Cyt aa3 [reduced, Fe(Ⅱ)] + Cyt c [oxidized, Fe(Ⅲ)]

- Half-reaction for reduction of oxygen is,

1/2 O2 + 2H+ + 2e- H2O

- Overall reaction is,

2Cyt aa3 [reduced, Fe(Ⅱ)] + 1/2 O2 + 2H+

2Cyt aa3 [oxidized, Fe(Ⅲ)] + H2O

- In this final reaction we have finally seen the link to molecular oxygen in aerobic metabolism.

- ΔG°’ = -220 kJ mol-1 for each mole of NADH


Complex Ⅳ

◈ Energetics of electron transport reactions.

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Cytochromes and Other Iron-Containing Proteins of Electron Transport

◈ All cytochromes contain heme group

- Differences in side chains of heme group: various stages of electron transport.


Cytochromes and Other Iron-Containing Proteins of Electron Transport

◈ Nonheme iron proteins

- Contain sulfur,

as case with iron-sulfur proteins

20.4 What Is the Connection between Electron Transport and Phosphorylation ?

◈ Some of energy released by electron transport is used for phosphorylation of ADP

- How each of reactions catalyzed by three of four respiratory complexes

① Energy-releasing oxidation reaction

② Proton pumping

③ pH gradient across inner mitochondrial membrane

④ Voltage difference across membrane by concentration difference of ions

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◈ Coupling factor (ATP synthase)

- To link oxidation and phosphorylation

- Complex protein oligomer

- F0: portion of protein that spans membrane, three polypeptide chains (a, b, and c)

- F1: portion that projects into matrix, five chains (α3β3γδε )


◈ ATP synthase

- The whole protein complex

- Mitochondrial ATPase


◈ Uncouplers

- Inhibit phosphorylation of ADP without affecting electron transport

- Ex) 2,4-dinitrophenol, various antibiotics (valinomycin, gramicidin A)


◈ P/O ratio

- To indicate coupling of ATP production to electron transport

- Number of moles of Pi consumed in the reaction ADP + Pi ATP for each mole of oxygen atoms consumed in the reaction

1/2O2 + 2H+ + 2e- H2O

- P/O ratio is 2.5 when NADH is substrate oxidized.

- P/O ratio is 1.5 when FADH2 is substrate oxidized.

20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?

Chemiosmotic Coupling

◈ Chemiosmotic coupling mechanism

- Difference in proton concentration between intermembrane space and matrix.

- By British scientist Peter Mitchell in 1961.



◈ Experimental evidence

1) A system with definite inside and outside compartments is essential for oxidative phosphorylation.

2) Submitochondrial preparations that contain closed vesicles can be prepared.



3) A model system for oxidative phosphorylation can be constructed with proton pumping in the absence of electron transport.

4) The existence of pH gradient has been

demonstrated and confirmed experimentally.



◈ How does proton gradient lead to production of ATP ?

- Ion channels through inner mitochondrial membrane



◈ Reasonable mode of action for uncouplers

- In light of the existence of a proton gradient

- Dinitrophenol (acid): Conjugate base is uncoupler.

- Ionophores: antibiotic uncouplers, such as gramicidin A and valinomycin


Conformational Aspects of Coupling

◈ Proton gradient is indirectly related to ATP production

- Proton gradient leads to conformational changes

in proteins.


Conformational Aspects of Coupling

- Three sites for substrate on synthase and three possible conformational sites

① Open (O), with low affinity for substrate

② Loose-binding (L), not catalytically active

③ Tight-binding (T), catalytically active

- F1 portion of ATP synthase as a rotary motor

- c, γ, and ε subunits constitute rotor.

- γ and ε subunits constitute rotating “shaft”.

20.6 How Are Respiratory Inhibitors Used to Study Electron Transport ?

◈ “If a pipeline is blocked, there will be a backup”

- In electron transport, this analogy is useful.

- When a flow of electrons is blocked in a series of redox reactions, reduced compounds will accumulate before blockage point in pathway.


◈ Respiratory inhibitors

- To determine order of electron transport chain

- Determining relative amounts of oxidized and reduced forms of various electron carriers in intact mitochondria

- On the spectroscopic properties


◈ Three sites at which inhibitors have an effect

1) Barbiturates block transfer of electrons

from flavoprotein NADH reductase

to coenzyme Q.

2) Blockage when electron transfer

involving b cytochromes, coenzyme Q,

and cytochrome c1

3) Transfer of electrons from

cytochrome aa3 complex to oxygen

20.7 What Are Shuttle Mechanisms ?

◈ Electrons can be transferred to a carrier that can cross membrane.

- NADH from glycolysis

to mitochondria

◈ Glycerol-phosphate shuttle

- Carrier system

- In insect flight muscle

- 1.5 ATP

20.7 What Are Shuttle Mechanisms ?

◈ Malate-aspartate shuttle

- More complex and more efficient shuttle mechanism

- In mammalian kidney, liver, and heart

- 2.5 ATP

20.8 What Is the ATP Yield from Complete Oxidation of Glucose ?

◈ How many molecules of ATP are produced for each molecule of glucose oxidized ?

- ATP production and reoxidation of NADH and FADH2.

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THE END!!

20. electron transport and oxidative phosphorylation -...

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