20. Electron Transport and Oxidative Phosphorylation -...
Transcript of 20. Electron Transport and Oxidative Phosphorylation -...
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20. Electron Transport and Oxidative Phosphorylation
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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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20.1 What Role Does Electron Transport Play in Metabolism ?
◈ 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
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20.1 What Role Does Electron Transport Play in Metabolism ?
◈ Oxygen, ultimate electron acceptor, is reduced to water
- Complete series of oxidation-reduction reactions
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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
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20.2 What Are the Reduction Potentials for the Electron Transport Chain ?
- Hydrogen/H+ pair has a higher reduction potential than ethanol/ acetaldehyde pair.
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20.2 What Are the Reduction Potentials for the Electron Transport Chain ?
- Electron flow in opposite direction (H2/H+ pair has a lower reduction potential.)
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20.2 What Are the Reduction Potentials for the Electron Transport Chain ?
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20.2 What Are the Reduction Potentials for the Electron Transport Chain ?
◈ 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
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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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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅰ
- Flavoprotein has a flavin coenzyme or FMN (flavin mononucleotide).
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20.3 How Are the Electron Transport Complexes Organized ?
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
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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅰ
- Overall equation
NADH + H+ + CoQ NAD+ + CoQH2
- Proton pumping create pH (proton) gradient
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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅰ
- Phosphorylation of ADP to ATP : ΔG°’ = -28.0 ~ -33.5 kJ mol-1.
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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅰ
◈ Some of carriers
- Carry electrons and hydrogens in reduced forms: NADH
- Carry only electrons: iron-sulfur protein
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20.3 How Are the Electron Transport Complexes Organized ?
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.
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20.3 How Are the Electron Transport Complexes Organized ?
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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20.3 How Are the Electron Transport Complexes Organized ?
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+
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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅲ
- CoQ can exist in three forms.
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20.3 How Are the Electron Transport Complexes Organized ?
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.
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20.3 How Are the Electron Transport Complexes Organized ?
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
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20.3 How Are the Electron Transport Complexes Organized ?
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
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20.3 How Are the Electron Transport Complexes Organized ?
Complex Ⅳ
◈ Energetics of electron transport reactions.
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20.3 How Are the Electron Transport Complexes Organized ?
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.
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20.3 How Are the Electron Transport Complexes Organized ?
Cytochromes and Other Iron-Containing Proteins of Electron Transport
◈ Nonheme iron proteins
- Contain sulfur,
as case with iron-sulfur proteins
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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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20.4 What Is the Connection between Electron Transport and Phosphorylation ?
◈ 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 )
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20.4 What Is the Connection between Electron Transport and Phosphorylation ?
◈ ATP synthase
- The whole protein complex
- Mitochondrial ATPase
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20.4 What Is the Connection between Electron Transport and Phosphorylation ?
◈ Uncouplers
- Inhibit phosphorylation of ADP without affecting electron transport
- Ex) 2,4-dinitrophenol, various antibiotics (valinomycin, gramicidin A)
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20.4 What Is the Connection between Electron Transport and Phosphorylation ?
◈ 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.
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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.
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
Chemiosmotic Coupling
◈ 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.
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
Chemiosmotic Coupling
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.
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
Chemiosmotic Coupling
◈ How does proton gradient lead to production of ATP ?
- Ion channels through inner mitochondrial membrane
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
Chemiosmotic Coupling
◈ 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
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
Conformational Aspects of Coupling
◈ Proton gradient is indirectly related to ATP production
- Proton gradient leads to conformational changes
in proteins.
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20.5 What Is the Mechanism of Coupling in Oxidative Phosphorylation ?
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”.
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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.
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20.6 How Are Respiratory Inhibitors Used to Study Electron Transport ?
◈ 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
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20.6 How Are Respiratory Inhibitors Used to Study Electron Transport ?
◈ 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
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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
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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
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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!!