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.

Role of Electron Transport in Metabolism

• 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

Figure 20.6 - Electron Transport Chain, Showing Respiratory Complexes

• 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

Figure 20.5 - Oxidized and Reduced Forms of Coenzyme Q

• 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:
  1. Food energy (glucose)
  2. NADH and FADH2
  3. Electron Transport Chain
  4. Oxygen
  5. 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–

Figure 20.11 - Iron-Sulfur Bonding in Nonheme Iron Proteins

• 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

Figure 20.13 - Model of the F1 and F0 Components of the ATP Synthase, a Rotating Molecular Motor

• 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

Figure 20.14 - Some Uncouplers of Oxidative Phosphorylation

• 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
  • Chemiosmotic coupling
• Depends on a conformational change in the ATP synthetase
  • Conformational coupling

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

Figure 20.15 - Creation of a Proton Gradient in Chemiosmotic Coupling

• 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

Figure 20.16 - Closed Vesicles

• Illustrates the concept of closed vesicles, essential for chemiosmotic coupling and oxidative phosphorylation.

Figure 20.17 - ATP Can Be Produced by Closed Vesicles with Bacteriorhodopsin as a Proton Pump

• 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

Figure 20.18 - Formation of ATP Accompanies the Flow of Protons Back into the Mitochondrial Matrix

• Illustrates the flow of protons back into the mitochondrial matrix through ATP synthase, driving ATP synthesis.

Conformational Coupling

• 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 Coupling (continued)

• 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

Figure 20.20 - The Glycerol-Phosphate Shuttle

• 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

Figure 20.21 - Malate-Aspartate Shuttle

• 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)