Biochemistry Notes: Electron Transport and Oxidative Phosphorylation
Electron Transport and Oxidative Phosphorylation
Chapter Outline
- The chapter covers the following topics:
- The role of electron transport in metabolism.
- Reduction potentials in the electron transport chain.
- Organization of electron transport complexes.
- The connection between electron transport and phosphorylation.
- The mechanism of coupling in oxidative phosphorylation.
- Shuttle mechanisms.
- The ATP yield from complete oxidation of glucose.
- In eukaryotic cells:
- Aerobic processes occur in the mitochondria.
- Glycolysis, an anaerobic process, occurs outside the mitochondria in the cytosol.
- Electron transport chain: Series of intermediate carriers that transfer electrons from NADH and FADH2 to O2.
- Reactions take place in the inner mitochondrial membrane.
Importance of Mitochondrial Structure in ATP Production
- Oxidative phosphorylation: Process for generating ATP.
- Depends on the creation of a pH gradient (proton gradient) within the mitochondrion as a result of electron transport.
- Proton gradient: Difference between the hydrogen ion concentrations in the mitochondrial matrix and that in the intermembrane space, which is the basis of coupling between oxidation and phosphorylation.
- Gives rise to most of the ATP production associated with the complete oxidation of glucose.
Mitochondria Structural Features
- Key structural features of mitochondria:
- Cristae: folds of the inner membrane
- Matrix: the space within the inner membrane
- Inner membrane
- Outer membrane
Establishment of a Proton Gradient
- Electron transport leads to proton pumping across the inner mitochondrial membrane.
- High [H+] = low pH in the intermembrane space.
- Low [H+] = high pH in the matrix.
Electron Transport Chain
- The electron transport chain involves the transfer of electrons from NADH and FADH2 to oxygen, coupled with the pumping of protons (H+) from the mitochondrial matrix to the intermembrane space, creating a proton gradient.
- Complexes I, II, III, and IV are involved in the electron transport chain.
- Coenzyme Q (CoQ) and cytochrome c (Cyt c) are mobile electron carriers within the electron transport chain.
- ATP synthase uses the proton gradient to synthesize ATP from ADP and inorganic phosphate (Pi).
Electron Transport
- Carried out by four closely related multienzyme systems and two electron carriers, coenzyme Q and cytochrome c
- Series of oxidation–reduction reactions in which NADH and FADH2 molecules generated in glycolysis and the citric acid cycle transfer electrons to oxygen
- NADH and FADH2 are oxidized to NAD+ and FAD
- O2 is reduced to H2O
- Results in protons being pumped across the inner membrane into the intermembrane space, creating a pH gradient
Electron Transport: Summary
- Electron transport from one carrier to another creates a proton gradient across the inner mitochondrial membrane
- Proton gradient is coupled to the production of ATP in aerobic metabolism
Reduction Potentials in the Electron Transport Chain
- A useful way to look at electron transport is to consider the change in free energy associated with the movement of electrons from one carrier to another
- If there are two electron carriers, such as NADH and coenzyme Q, are electrons more likely to be transferred from NADH to coenzyme Q or vice versa?
- This is determined by measuring a reduction potential for the carriers
- Molecule with a high reduction potential tends to be reduced if it is paired with a molecule with a lower reduction potential
Measuring Reduction Potentials
- Reference point is the half-cell on the right where H+ is in aqueous solution in equilibrium with hydrogen gas
- Reduction of H+ to hydrogen gas is considered to have a voltage (E) of zero
- Sample to be tested is in the other half-cell
Table 20.1 - Standard Reduction Potentials for Several Biological Reduction Half Reactions
- The table lists standard reduction potentials (E°') for various biological redox half-reactions.
- It includes reactions involving oxygen, iron, cytochromes, coenzyme Q, fumarate, succinate, FAD/FADH2, oxaloacetate, malate, pyruvate, lactate, acetaldehyde, ethanol, FMN/FMNH2, NAD+/NADH, NADP+/NADPH, α-Ketoglutarate, Isocitrate and Succinate.
Calculation of \Delta G°'
- \Delta G°' represents the free-energy change of a reaction under standard conditions
- \Delta G°' of a redox reaction is calculated using the following equation:
- n - Number of moles of electrons transferred
- F - Faraday’s constant (96.485 \text{ kJ V}^{-1} \text{mol}^{-1})
- \Delta E°' - Total voltage for the two half reactions
- \Delta G°' = -nF \Delta E°'
Reduction Potentials: Summary
- Standard reduction potentials:
- Provide a basis for comparison among redox reactions
- Help predict sequence of reactions in the electron transport chain
- Complex I: NADH-CoQ oxidoreductase
- Complex II: Succinate-CoQ oxidoreductase
- Complex III: CoQH-cytochrome c oxidoreductase
- Complex IV: Cytochrome oxidase
Complex I
- NADH-CoQ oxidoreductase
- Catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ)
- Integral part of the inner mitochondrial membrane
- Includes several proteins that contain an iron–sulfur cluster and the flavoprotein that oxidizes NADH
- Flavoprotein has a flavin coenzyme called flavin mononucleotide (FMN)
Complex I (continued 1)
- Stage 1 - Transfer of electrons from NADH to the flavin portion of the flavoprotein
- Stage 2 - Reduced FMN is reoxidized, and the oxidized form of the iron–sulfur protein is reduced
- Stage 3 - Reduced iron–sulfur protein donates its electrons to CoQ, which is reduced to CoQH2
- \text{NADH} + \text{H}^+ + \text{E-FMN} \rightarrow \text{NAD}^+ + \text{E-FMNH}_2
- \text{E-FMNH}2 + 2 \text{Fe-S}{ \text{oxidized} } \rightarrow \text{E-FMN} + 2 \text{Fe-S}_{ \text{reduced} } + 2\text{H}^+
- 2 \text{Fe-S}{ \text{reduced} } + \text{CoQ} + 2 \text{H}^+ \rightarrow 2 \text{Fe-S}{ \text{oxidized} } + \text{CoQH}_2
- Coenzyme Q (CoQ) exists in two primary forms:
- Oxidized quinone form (CoQ)
- Reduced hydroquinone form (CoQH2)
Complex I (continued 2)
- Overall equation for the reaction is given as follows:
- \text{NADH} + \text{H}^+ + \text{CoQ} \rightarrow \text{NAD}^+ + \text{CoQH}_2
- \Delta G°' indicates that the reaction is strongly exergonic and releases enough energy to drive the phosphorylation of ADP to ATP
Complex II
- Succinate-coenzyme Q oxidoreductase catalyzes the transfer of electrons from succinate to CoQ
- Stage 1 - Succinate is oxidized to fumarate by a flavin enzyme
- E—FAD indicates that the flavin portion is covalently bonded to the enzyme
- Stage 2 - Flavin group is reoxidized as another iron–sulfur protein is reduced
- \text{Succinate} + \text{E-FAD} \rightarrow \text{Fumarate} + \text{E-FADH}_2
- \text{E-FADH}2 + \text{Fe-S}{ \text{oxidized} } \rightarrow \text{E-FAD} + \text{Fe-S}_{ \text{reduced} }
Complex II (continued)
- Stage 3 - Reduced iron–sulfur protein donates its electrons to oxidized CoQ, and CoQ is reduced
- Overall reaction
- Exergonic (\Delta G°' = -13.5 \text{ kJ mol}^{-1})
- Energy from this reaction is not enough to drive ATP production
- No H+ is pumped out of the matrix during this step
- \text{Fe-S}{ \text{reduced} } + \text{CoQ} + 2 \text{H}^+ \rightarrow \text{Fe-S}{ \text{oxidized} } + \text{CoQH}_2
- \text{Succinate} + \text{CoQ} \rightarrow \text{Fumarate} + \text{CoQH}_2
Action of Cytochromes
- Electrons are passed from CoQ, which is reoxidized, to cytochromes
- Cytochromes: Groups of heme-containing proteins in the electron transport chain
- In each heme group, the iron is successively reduced to Fe (II) and reoxidized to Fe (III)
- Different types of cytochromes are distinguished by lowercase letters (a, b, c), and further distinction is made possible with subscripts (c1)
- \text{Fe(III)} + e^- \rightarrow \text{Fe(II)} \text{ (reduction)}
- \text{Fe(II)} \rightarrow \text{Fe(III)} + e^- \text{ (oxidation)}
Heme Group of Cytochromes
- All cytochromes contain a heme group
- There are differences in the side chain depending on the heme
Complex III
- CoQH2-cytochrome c oxidoreductase catalyzes the oxidation of reduced coenzyme Q
- Electrons produced by this reaction are passed along to cytochrome c in a multistep process
- H+ ions pass out on the other side of the membrane
- Overall reaction
- Two molecules of cytochrome c are required for every molecule of coenzyme Q
- Components include two b-type cytochromes (bH and bL), cytochrome c1, and several iron–sulfur proteins
- \text{CoQH}_2 + 2 \text{Cyt } c \text{ (Fe(III))} \rightarrow \text{CoQ} + 2 \text{Cyt } c \text{ (Fe(II))} + 2\text{H}^+
Complex III (continued)
- Integral part of the inner mitochondrial membrane
Complex III: Q Cycle
- Series of reactions in the electron transport chain
- Provides the link between two-electron transfers and one-electron transfers
- Involves the flow of electrons via a cyclic path from CoQH2 to other components of the complex
- Depends on the fact that coenzyme Q can exist in three forms
Complex III: Q Cycle (continued 1)
- One electron is passed from CoQH2 to the iron–sulfur clusters to cytochrome c1, leaving coenzyme Q in the semiquinone form
- Semiquinone, along with the oxidized and reduced forms of coenzyme Q, participates in a cyclic process in which two b cytochromes are reduced and oxidized in turn
- Each molecule of coenzyme Q loses one electron
- \text{CoQH}2 \rightarrow \text{Fe-S} \rightarrow \text{Cyt } c1
- \text{CoQH} + \text{Cyt } c \text{ (oxidized)} \rightarrow \text{Cyt } c \text{ (reduced)} + \text{CoQ semiquinone anion} + 2 \text{H}^+
Complex III: Q Cycle (continued 2)
- Provides a mechanism for electrons to be transferred one at a time from coenzyme Q to cytochrome c1
- Complex III results in proton pumping and supplies enough energy to drive ATP production because of the reaction that it catalyzes
Complex IV
- Cytochrome c oxidase catalyzes the transfer of electrons from cytochrome c to O2
- Overall reaction
- Results in proton pumping
- Integral part of the inner mitochondrial membrane and contains cytochromes a and a3, as well as two Cu2+ ions that are involved in electron transport
- 2 \text{Cyt } c \text{ (Fe(II))} + 2\text{H}^+ + \frac{1}{2} \text{O}2 \rightarrow 2 \text{Cyt } c \text{ (Fe(III))} + \text{H}2\text{O}
Complex IV (continued)
- Cu2+ ions are intermediate electron acceptors that lie between cytochromes a and a3 in the following sequence:
- \text{Cyt } c \rightarrow \text{Cyt } a \rightarrow \text{Cu} \rightarrow \text{Cyt } a3 \rightarrow \text{O}2
- Reaction of the cytochromes:
- \text{Cyt } a3 \text{ (reduced, Fe(II))} + \text{Cyt } a \text{ (oxidized, Fe(III))} \rightarrow \text{Cyt } a3 \text{ (oxidized, Fe(III))} + \text{Cyt } a \text{ (reduced, Fe(II))}
- 2 \text{Cyt } a \text{ (reduced, Fe(II))} + \text{O}2 + 2\text{H}^+ \rightarrow 2 \text{Cyt } a \text{ (oxidized, Fe(III))} + \text{H}2\text{O}
Table 20.2 - Energetics of Electron Transport Reactions
- The table provides the free energy changes involved in electron transport reactions.
- Lists \Delta G°' in kJ/mol NADH and kcal/mol NADH for individual steps in the electron transport chain. For example:
- NADH + H+ + E-FMN → NAD+ + E—FMNH₂ : \Delta G°' = -38.6 \text{ kJ (mol NADH)}^{-1} or -9.2 \text{ kcal (mol NADH)}^{-1}
- Overall reaction: NADH + H+ + ½ O2 → NAD+ + H₂O : \Delta G°' = -220 \text{ kJ (mol NADH)}^{-1} or -52.6 \text{ kcal (mol NADH)}^{-1}
Example 20-1
- Draw an Energy Diagram for our Electron Transport Chain Reactions
- The overall reaction is to split an oxygen molecule and form water; why can’t we do this in one simple step????
Solution 20-1
- Lists Steps:
- Food energy (glucose)
- NADH and FADH2
- Electron Transport Chain
- Oxygen
- H2O
- ATP produced at each step.
- -\Delta G at each step.
Solution 20-1 Cont’d
- The reaction of H2 + ½ O2 -> H2O can proceed two ways, but both methods will require a potential of -600 mVolts to -1200 mVolts. Your cell membrane resting potentials are roughly < -100 mVolts.
- Generating such a high voltage all at once will be a BIG problem for your neurons etc.
Redox Role of Heme in Cytochromes
- All cytochromes involved in various stages of the electron transport chain contain heme groups that differ in properties
- Iron of the heme group is involved in a series of redox reactions
- Side chains of heme groups are different
- Variations exist in the polypeptide chain and in the way the polypeptide chain is attached to the heme
- Nonheme iron proteins contain sulfur
- Iron is usually bound to cysteine or to S2–
- Illustrates iron-sulfur clusters in nonheme iron proteins, where iron is bound to cysteine residues and sulfide ions.
Connection between Electron Transport and Phosphorylation
- Energy-releasing oxidation reactions give rise to proton pumping and a pH gradient across the inner mitochondrial membrane, which is used to drive the phosphorylation of ADP
- Differences in the concentration of ions across the membrane generate a voltage gradient
- Coupling process converts the energy of the electrochemical potential (voltage drop) across the membrane to the chemical energy of ATP
Coupling Factor
- Needed to link oxidation and phosphorylation
- ATP synthase (mitochondrial ATPase): Complex protein oligomer that is responsible for the production of ATP in the mitochondria
- F0 - Portion of the protein that spans the membrane
- Consists of three different kinds of polypeptide chains (a, b, and c)
- F1 - Portion of the protein that projects into the matrix
- Consists of five different kinds of polypeptide chains in the ratio \alpha3 \beta3 \gamma \delta \epsilon
- Site of ATP synthesis
- Illustrates the structure of ATP synthase, highlighting the F1 and F0 components and their roles in ATP synthesis.
Uncouplers
- Inhibit the phosphorylation of ADP without affecting electron transport
- Reduce oxygen to H2O but do not enable the production of ATP
- Examples
- 2,4-dinitrophenol
- Valinomycin
- Gramicidin A
- 2,4-Dinitrophenol (DNP)
- Valinomycin
- Gramicidin A
P/O Ratio
- Ratio of ATP produced by oxidative phosphorylation to oxygen atoms consumed in electron transport
- P/O = 2.5 when NADH is oxidized
- P/O = 1.5 when FADH2 is oxidized
- Phosphorylation: ADP + P_{i} → ATP
- Oxidation: O2 + 4H^+ + 4e^- → 2H2O
Connection between Phosphorylation and Electron Transport: Summary
- Coupling of electron transport to oxidative phosphorylation requires the complex protein oligomer, ATP synthase
- This enzyme has a channel for protons to flow from the intermembrane space into the mitochondrial matrix
- Proton flow is coupled to ATP production in a process that appears to involve a conformational change of the enzyme
Mechanisms of Coupling in Oxidative Phosphorylation
- Requires a proton gradient across the inner mitochondrial membrane
- Depends on a conformational change in the ATP synthetase
Chemiosmotic Coupling
- Proton gradient exists because the various proteins that serve as electron carriers:
- Are not symmetrically oriented with respect to the two sides of the inner mitochondrial membrane
- Do not react in the same way with respect to the matrix and the intermembrane space
- Proteins that serve as electron carriers take up protons from the matrix when they are reduced and release them to the intermembrane space when they are reoxidized
- Reactions of NADH, CoQ, and O2 require protons
- Illustrates the mechanism of chemiosmotic coupling, showing electron flow, proton pumping, and the creation of a proton gradient across the inner mitochondrial membrane.
Evidence Supporting Chemiosmotic Coupling (Peter Mitchell, 1961)
- System with definite inside and outside compartments (closed vesicles) is essential for oxidative phosphorylation
- Submitochondrial preparations that contain closed vesicles can be produced
- Such vesicles can carry out oxidative phosphorylation
- Asymmetric orientation of respiratory complexes with respect to the membrane can be demonstrated
Evidence Supporting Chemiosmotic Coupling (Peter Mitchell, 1961) (continued)
- Model system for oxidative phosphorylation can be constructed with proton pumping in the absence of electron transport
- System consists of reconstituted membrane vesicles, mitochondrial ATP synthase, and a proton pump (bacteriorhodopsin)
- Existence of the pH gradient has been demonstrated and confirmed experimentally
- Illustrates the concept of closed vesicles, essential for chemiosmotic coupling and oxidative phosphorylation.
- Demonstrates that ATP can be produced by closed vesicles with bacteriorhodopsin as a proton pump, supporting the chemiosmotic coupling theory.
Chemiosmotic Coupling: Flow of Protons
- Way by which the proton gradient leads to the production of ATP depends on ion channels through the inner mitochondrial membrane
- Protons flow back into the matrix through channels in the F0 unit of ATP synthase
- Flow of protons is accompanied by formation of ATP, which takes place in the F1 unit
- Details of how phosphorylation takes place as a result of the linkage to the proton gradient are not explicitly specified by this mechanism
- Illustrates the flow of protons back into the mitochondrial matrix through ATP synthase, driving ATP synthesis.
- Proton gradient leads to conformational changes in a number of proteins, including ATP synthase
- There are three sites for substrate on ATP synthase and three possible conformational states
- Open (O)
- Low affinity for substrate
- Loose-binding (L)
- Not catalytically active
- ADP and Pi bind at a site in this conformation
- Tight-binding (T)
- Catalytically active
- ATP is bound at a site in this conformation
- Conformational states interconvert as a result of proton flux through the synthase that converts:
- T to O, releasing ATP
- L to T, which produces ATP
Coupling Mechanisms: Summary
- In chemiosmotic coupling, the proton gradient is the crux of the matter
- Flow of protons through the pore in the synthase drives ATP production
- In conformational coupling, a change in the shape of the ATP synthase releases bound ATP that has already been formed
Shuttle Mechanisms
- Transport metabolites between the mitochondria and the cytosol
- Glycerol–phosphate shuttle: Mechanism for transferring electrons from NADH in the cytosol to FADH2 in the mitochondrion
- Glycerol phosphate is produced by the reduction of dihydroxyacetone phosphate
- NADH is oxidized to NAD+, and FAD is the oxidizing agent
- FADH2 is the product, which passes electrons through the electron transport chain
- 1.5 moles of ATP are produced for each cytosolic NADH
- Illustrates the glycerol-phosphate shuttle, a mechanism for transferring electrons from cytosolic NADH to mitochondrial FADH2.
Shuttle Mechanisms (continued)
- Malate–aspartate shuttle
- Has been found in mammalian kidney, liver, and heart
- Uses the fact that malate can cross the mitochondrial membrane, while oxaloacetate cannot
- Transfer of electrons from NADH in the cytosol produces NADH in the mitochondrion
- 2.5 moles of ATP are produced for each mole of cytosolic NADH
Malate–Aspartate Shuttle
- In the cytosol:
- Oxaloacetate is reduced to malate by cytosolic malate dehydrogenase
- Cytosolic NADH is oxidized to NAD+
- Malate crosses the mitochondrial membrane
- In the mitochondrion:
- Conversion of malate back to oxaloacetate is catalyzed by mitochondrial malate dehydrogenase
- Oxaloacetate is converted to aspartate, which can cross the mitochondrial membrane
- Aspartate is converted to oxaloacetate in the cytosol
- Illustrates the malate-aspartate shuttle, a mechanism for transferring electrons from cytosolic NADH to mitochondrial NADH.
Shuttle Mechanisms: Summary
- Shuttle mechanisms transfer electrons, but not NADH, from the cytosol across the mitochondrial membrane
- In the malate–aspartate shuttle, 2.5 moles of ATP are produced for each molecule of cytosolic NADH, rather than 1.5 moles of ATP in the glycerol–phosphate shuttle, a point that affects the overall yield of ATP in these tissues
ATP Yield from Complete Oxidation of Glucose
- Total of 30 or 32 ATPs are produced when:
- Pyruvate generated from glycolysis can enter the citric acid cycle
- NADH and FADH2 molecules that result from the citric acid cycle are reoxidized through the electron transport chain
Table 20.3 - Yield of ATP from Glucose Oxidation
- Details the ATP yield per glucose molecule from glycolysis, pyruvate conversion to acetyl-CoA, and the citric acid cycle.
- Shows differences based on whether the glycerol-phosphate shuttle (30 ATP) or the malate-aspartate shuttle (32 ATP) is used.
- ATP Yield per Glucose:
- Net yield: +30 (+32 w/ Malate-Aspartate Shuttle)