1. Chapter 10
Chemotrophic Energy Metabolism: Aerobic Respiration

2. Chemotrophic Energy Metabolism: Aerobic Respiration
Some cells meet their energy needs through anaerobic fermentation
However, fermentation yields only modest amounts of energy due to the absence of electron transfer
ATP yield is much higher in cellular respiration 2

3. Cellular Respiration: Maximizing ATP Yields
Cellular respiration (or respiration) uses an external electron acceptor to oxidize substrates completely to CO2
External electron acceptor: one that is not a by-product of glucose catabolism

4. Cellular respiration defined
Respiration is the flow of electrons through or within a membrane, from reduced coenzymes to an external electron acceptor usually accompanied by the generation of ATP
Coenzymes such as FAD (flavin adenine dinucleotide) and coenzyme Q (ubiquinone) are involved

5. The terminal electron acceptor
In aerobic respiration, the terminal electron acceptor is oxygen and the reduced form is water
Other terminal electron acceptors (sulfur, protons, and ferric ions) are used by other organisms, especially bacteria and archaea
These are examples of anaerobic respiration

6. Mitochondria
Most aerobic ATP production in eukaryotic cells takes place in the mitochondrion
In bacteria, the plasma membrane and cyotoplasm are analogous to the mitochondrial inner membrane and matrix with respect to energy metabolism

7. Figure 10-1

8. Bioflix: Cellular Respiration

9. Aerobic Respiration Yields Much More Energy than Fermentation Does
With O2 as the terminal electron acceptor, pyruvate can be oxidized completely to CO2
Aerobic respiration has the potential of generating up to 38 ATP molecules per glucose
Oxygen provides a means of continuous reoxidation of NADH and other reduced coenzymes

10. Respiration Includes Glycolysis, Pyruvate Oxidation, the TCA Cycle, Electron Transport, and ATP Synthesis
Respiration will be considered in five stages
Stage 1: the glycolytic pathway
Stage 2: pyruvate is oxidized to generate acetyl CoA
Stage 3: acetyl Co A enters the tricarboxylic acid cycle (TCA cycle), where it is completely oxidized to CO2

11. The five stages, continued
Respiration will be considered in five stages
Stage 4: electron transport, the transfer of electrons from reduced coenzymes to oxygen coupled to active transport of protons across a membrane
Stage 5: The electrochemical proton gradient formed in step 4 is used to drive ATP synthesis (oxidative phosphorylation)

12. The Mitochondrion: Where the Action Takes Place
The mitochondrion is called the “energy powerhouse” of the eukaryotic cell
These organelles are thought to have arisen from bacterial cells
Mitochondria have been shown to carry out all the reactions of the TCA cycle, electron transport, and oxidative phosphorylation

13. Mitochondria Are Often Present Where the ATP Needs Are Greatest
Mitochondria are found in virtually all aerobic cells of eukaryotes
They are present in both chemotrophic and phototrophic cells
Mitochondria are frequently clustered in regions of cells with the greatest need for ATP, e.g., muscle cells

14. Figure 10-2

15. Are Mitochondria Interconnected Networks Rather than Discrete Organelles?
In electron micrographs, mitochondria usually appear as oval structures
However they can take various shapes and sizes, depending on the cell type
Their appearance under EM suggests that they are large, and numerous discrete entities

16. A challenge to the accepted view
The work of Hoffman and Avers suggests that the oval profiles in electron micrographs of yeast cells reflect slices through a single large branched mitochondrion
This work suggests that mitochondria may be larger than previously thought, and less numerous
There is additional support for this idea

17. The Outer and Inner Membranes Define Two Separate Compartments and Three Regions
A distinctive feature of mitochondria is the presence of both outer and inner membranes
The outer membrane contains porins that allow passage of solutes with molecular weights up to 5000
The intermembrane space between the inner and outer membranes is thus continuous with the cytosol

18. The inner membrane
The inner membrane of the mitochondria is impermeable to most solutes, partitioning the mitochondrion into two separate compartments
The intermembrane space
The interior of the organelle, or mitochondrial matrix

19. Figure 10-3

20. Figure 10-3A

21. Figure 10-3B

22. Video: Mitochondria in 3-D

23. Figure 10-4

24. The inner boundary membrane and cristae
The portion of the inner membrane adjacent to the intermembrane space is called the inner boundary membrane
The inner membrane is about 75% protein by weight; the proteins include those involved in solute transport, electron transport, and ATP synthesis

25. The cristae
The inner membrane of most mitochondria has many infoldings called cristae
They increase surface area of the inner membrane, and provide more space for electron transport to take place
The cristae provide localized regions, intracristal spaces, where protons can accumulate during electron transport

26. The cristae (continued)
The cristae are thought to be tubular structures that associate in layers
They have limited connections to the inner boundary membrane through small openings, crista junctions
Cells with high metabolic activity seem to have more cristae in their mitochondria

27. The mitochondrial matrix
The interior of the mitochondrion is filled with a semi-fluid matrix
The matrix contains many enzymes involved in mitochondrial function as well as DNA molecules and ribosomes
Mitochondria contain proteins encoded by their own DNA as well as some that are encoded by nuclear genes

28. Mitochondrial Functions Occur in or on Specific Membranes and Compartments
Specific functions and pathways have been localized within mitochondria by fractionation studies
Most of the enzymes involved in pyruvate oxidation, the TCA cycle, and catabolism of fatty acids and amino acids are found in the matrix
Most electron transport intermediates are integral inner membrane components

29. Table 10-1

30. Localization of Specific Mitochondrial Functions
Knoblike spheres called F1 complexes protrude from the inner membrane into the matrix
These are involved in ATP synthesis
Each complex is an assembly of several different polypeptides, and can be seen in an electron micrograph using negative staining

31. Figure 10-5

32. Figure 10-5A

33. Figure 10-5B

34. Figure 10-5C

35. F1 complexes
Each F1 complex is attached by a short protein stalk to an Fo complex
This is an assembly of hydrophobic polypeptides embedded in the mitochondrial inner membrane
This FoF1 complex is an ATP synthase that is responsible for most of the ATP generation in the mitochondria (and in bacterial cells as well)

36. In Bacteria, Respiratory Functions Are Localized to the Plasma Membrane and the Cytoplasm
Bacteria do not have mitochondria but are capable of aerobic respiration
Their plasma membrane and cytoplasm perform the same functions as the inner membrane and matrix of mitochondria

37. The Tricarboxylic Acid Cycle: Oxidation in the Round
In the presence of oxygen pyruvate is oxidized fully to carbon dioxide with the released energy used to drive ATP synthesis
This involves the TCA (tricarboxylic acid) cycle, in which citrate is an important intermediate
The TCA cycle is also called the Krebs cycle after Hans Krebs, whose lab played a key role in elucidating the cycle

38. The Tricarboxylic Acid Cycle
The TCA cycle metabolized acetyl CoA, produced from pyruvate decarboxylation
Acetyl CoA can also arise from fatty acid oxidation
Acetyl CoA transfers its acetate group to a four-carbon acceptor called oxaloacetate, generating citrate

39. The fate of citrate
After its formation, citrate undergoes two successive decarboxylations
It also goes through several oxidation steps
Eventually oxaloacetate is regenerated, and can accept two more carbons from acetyl CoA and the cycle begins again

40. The overall cycle
Each round of the TCA cycle involves the entry of two carbons, the release of two CO2, and the regeneration of oxaloacetate
Oxidation occurs at five steps, four in the cycle itself and one when pyruvate is converted to acetyl CoA
In each case, electrons are accepted by coenzymes

41. Figure 10-6

42. Pyruvate Is Converted to Acetyl Coenzyme A by Oxidative Decarboxylation
The glycolytic pathway ends with pyruvate, which is small enough to enter the intermembrane space of the mitochondrion
At the inner mitochondrial membrane, a specific symporter transports pyruvate into the matrix, along with a proton
Then, pyruvate is converted to acetyl CoA by pyruvate dehydrogenase complex (PDH)

43. Conversion of pyruvate
The conversion is a decarboxylation because one carbon is liberated as CO2
It is also an oxidation because two electrons (and one proton) are transferred to NAD+ to form NADH
Coenzyme A contains the B vitamin pantothenic acid
Coenzyme A has a SH group that makes it a good carrier of acetate (and other organic acids)

44. Conversion of pyruvate
The conversion of pyruvate to acetyl CoA can be summarized as follows:

45. Figure 10-7

46. The TCA Cycle Begins with the Entry of Acetate as Acetyl CoA
With each round of the TCA cycle, two carbon atoms enter in organic form as acetate and leave in inorganic form as carbon dioxide
In the first reaction, TCA-1, the two-carbon acetate group is transferred from acetyl CoA to oxaloacetate (4C) to form citrate (6C)
This reaction is catalyzed by citrate synthetase

47. TCA-2
Citrate is converted to isocitrate in the second step of the TCA cycle
The enzyme aconitase catalyzes the reaction
Isocitrate has a hydroxyl group that is easily oxidized (or dehydrogenated) in step TCA-3

48. Figure 10-8

49. Two Oxidative Decarboxylations Then Form NADH and Release CO2
Four of the eight steps in the TCA cycle are oxidations (TCA-3, TCA-4, TCA-6, TCA-8)
These all involve coenzymes that enter in the oxidized form and leave in the reduced form
The first two of these are decarboxylation steps, releasing one CO2 in each step

50. TCA-3, TCA-4
Isocitrate is oxidized by isocitrate dehydrogenase to oxalosuccinate, with NAD+ as the electron acceptor
Oxalosuccinate immediately undergoes decarboxylation to form a-ketoglutarate (5C)(TCA-3)
a-ketoglutarate is oxidized to succinyl CoA, by a-ketoglutarate dehydrogenase (TCA-4)

51. Direct Generation of GTP (or ATP) Occurs at One Step in the TCA Cycle
So far in the cycle, two carbon atoms have entered and two have left (but not the same two carbons), and two molecules of NADH have been generated
Succinyl CoA has been generated; like acetyl CoA it has a high-energy thioester bond
The energy from hydrolysis of this bond is used to generate one ATP (bacteria, plants) or GTP(animals) (TCA-5)

52. The Final Oxidative Reactions of the TCA Cycle Generate FADH2 and NADH
Of the remaining three steps in the cycle, two are oxidations
Succinate is oxidized to fumarate (TCA-6); this transfers electrons to FAD, a lower-energy coenzyme than NAD+
In the next step, fumarate is hydrated to produce malate (TCA-7) by fumarate hydratase

53. Figure 10-9

54. TCA-8
In the final step of the TCA cycle, the hydroxyl group of malate becomes the target of the final oxidation in the cycle
Electrons are transferred to NAD+, producing NADH as malate is converted to oxaloacetate (TCA-8)

55. Summing Up: The Products of the TCA Cycle Are CO2, ATP, NADH, and FADH2
The TCA cycle accomplishes the following:
1. Two carbons enter the cycle as acetyl CoA, which joins oxaloacetate to form the six-carbon citrate
2. Decarboxylation occurs at two steps to balance the input of two carbons by releasing two CO2
3. Oxidation occurs at four steps, with NAD+ the electron acceptor in three steps and FAD in one

56. The Products of the TCA Cycle Are CO2, ATP, NADH, and FADH2 (continued)
The TCA cycle accomplishes the following:
4. ATP is generated at one point, with GTP as an intermediate in the case of animal cells
5. One turn of the cycle is completed as oxaloacetate, the original 4C acceptor, is regenerated

57. Summing up the TCA cycle
The TCA cycle can be summarized as follows:
Including glycolysis, pyruvate decarboxylation, and the TCA cycle

58. Several TCA Cycle Enzymes Are Subject to Allosteric Regulation
Like all metabolic pathways, the TCA cycle must be carefully regulated to meet cellular needs
Most of the control of the cycle involves allosteric regulation of four key enzymes by specific effector molecules
Effector molecules may be activators or inhibitors 58

59. Regulation of PDH
PDH is reversibly inactivated by phosphorylation and activated by dephosphorylation of one of its protein components
PDH is inhibited by ATP, which is abundant where energy is plentiful
PDH and isocitrate dehydrogenase are activated by AMP and ADP, which are more abundant when energy is needed 59

60. Regulation of acetyl CoA
Overall availability of acetyl CoA is determined mainly by the activity of the PDH complex
PDH is allosterically inhibited by ATP, NADH, and acetyl CoA and high [ATP/ADP] (enzyme: PDH kinase)
It is activated by AMP, NAD+, and free CoA and low [ATP/ADP] (enzyme: PDH phosphatase) 60

61. Figure 10-10

62. The TCA Cycle Also Plays a Central Role in the Catabolism of Fats and Proteins
The TCA cycle represents the main conduit of aerobic energy metabolism for a variety of substrates besides sugar, in particular, fats and proteins

63. Fat as a Source of Energy
Fats are highly reduced compounds that liberate more energy per gram upon oxidation than do carbohydrates
They are a long-term energy storage form for many organisms
Most fat is stored as deposits of triacylglycerols, neutral triesters of glycerol and long-chain fatty acids

64. Catabolism of triacylglycerols
Triacylglycerol catabolism begins with their hydrolysis to glycerol and free fatty acids
The glycerol is channeled into the glycolytic pathway by oxidative conversion to dihydroxyacetone phosphate
Fatty acids are linked to coenzyme A, to form fatty acyl CoAs, then degraded by b-oxidation

65. b-oxidation
b-oxidation is a catabolic process that generates acetyl CoA and the reduced coenzymes NADH and FADH2
b-oxidation occurs in different compartments in different organisms
Here, we will focus on the mitochondrion of animals using saturated fatty acids with an even number of carbons as an energy source

66. Fatty acid degradation
Most fatty acids are oxidatively converted to acetyl CoA in the mitochondrion
These can be further catabolized in the TCA cycle
The fatty acids are degraded in a series of repetitive cycles, which removes two carbons at a time until the fatty acid is completely degraded

67. Steps of b-oxidation Each cycle involves
2. Hydration
3. Reoxidation
4. Thiolysis
The result is the production of one FADH2, one NADH, and one acetyl CoA per cycle

68. Formation of fatty acyl CoA
b-oxidation of a fatty acid begins with an activation step in the cytosol that requires the energy of ATP hydrolysis
This drives the attachment of a CoA molecule to the fatty acid (FA-1)
The fatty acyl CoA is then transported into the mitochondrion by a translocase in the inner membrane

69. Degradation of fatty acyl CoA
An integral membrane dehydrogenase oxidizes the fatty acyl CoA, forming a double bond between the a and b carbons (FA-2)
The two electrons and protons removed are transferred to FAD, forming FADH2
Water is added across the double bond by a hydratase (FA-3)

70. Figure 10-11

71. Degradation of fatty acyl CoA (continued)
Another dehydrogenase oxidizes the b-carbon, converting the hydroxyl group to a keto group (FA-4)
Two electrons and one proton are transferred to NAD+ to form NADH
The bond between the a and b carbons is broken by a thiolase and a two-carbon fragment is transferred to a second acetyl CoA (FA-5)

72. Degradation of fatty acyl CoA (continued)
The steps FA-2 to FA-5 are repeated until the original fatty acid is completely degraded
Most fatty acids have an even number of carbons and are completely degraded; those with an uneven number of carbons require extra steps before feeding into the TCA cycle
Unsaturated fatty acids require one or two additional enzymes

73. Protein as a Source of Energy and Amino Acids
Besides their other numerous functions, proteins can be catabolized to produce ATP if necessary when carbohydrate and lipid stores are depleted
Eventually cells undergo turnover of proteins and protein-containing structures
The resulting amino acids can be used to generate new proteins or degraded for energy

74. Protein catabolism
Protein catabolism begins with hydrolysis of the peptide bonds that link amino acids together
This is called proteolysis and the enzymes responsible for it are called proteases
The products of proteolysis are small peptides and free amino acids

75. Endopeptidases and exopeptidases
Further digestion of peptides is catalyzed by peptidases
Endopeptidases hydrolyze internal peptide bonds
Exopeptidases remove successive amino acids from the end of the peptide

76. Amino acid catabolism Free amino acids can be catabolized for energy
These are converted into intermediates of mainstream catabolism in as few steps as possible
The pathways differ for individual amino acids, but all eventually lead to acetyl CoA, pyruvate, or a few key TCA cycle intermediates

77. Figure 10-12

78. The TCA Cycle Serves as a Source of Precursors for Anabolic Pathways
In addition to the catabolic function of the TCA cycle, there is a flow of four-, five- and six-carbon intermediates into and out of it in most cells
These side reactions can use intermediates for the synthesis of needed compounds
The TCA cycle is a link between anabolic and catabolic pathways and is therefore amphibolic

79. The TCA cycle has a central role in some anabolic processes
a-keto intermediates of the TCA cycle can be converted into the amino acids alanine, aspartate, and glutamate
Succinyl CoA is the starting point for heme synthesis
Citrate can be transported out of the mitochondrion and used as an acetyl CoA source for fatty acid synthesis

80. The Glyoxylate Cycle Converts Acetyl CoA to Carbohydrates
The glyoxylate cycle has several intermediates in common with the TCA cycle but performs a specialized function in some plant cells and fungi
This occurs in a specialized peroxisome called the glyoxosome
Two acetate molecules (from acetyl CoA) form succinate which is converted to PEP, from which sugars can be synthesized

81. Figure 10-10A-1 81

82. Figure 10-10A-2 82

83. Electron Transport: Electron Flow from Coenzymes to Oxygen
Chemotrophic energy metabolism through the TCA cycle accounts for synthesis of 4 ATP per glucose (2 from glycolysis and 2 from the TCA cycle)
This accounts for only a small portion of the energy in the original glucose molecule
The remainder is stored in NADH and FADH2

84. The Electron Transport System Conveys Electrons from Reduced Coenzymes to Oxygen
Coenzyme reoxidation by transfer of electrons to oxygen is called electron transport
Electron transport and ATP generation are not independent processes; they are functionally linked to each other

85. Electron Transport and Coenzyme Oxidation
Electron transport involves the highly exergonic oxidation of NADH and FADH2 with O2 as the terminal electron acceptor and so accounts for the formation of water

86. The Electron Transport System
Electron transfer is carried out as a multistep process involving an ordered series of reversibly oxidized electron carriers functioning together
This is called the electron transport system, ETS
The ETS contains a number of integral membrane proteins that are found in the inner mitochondrial membrane (or plasma membrane of bacteria)

87. The Electron Transport System Consists of Five Kinds of Carriers
Flavoproteins
Iron-sulfur proteins
Cytochromes
Copper-containing cytochromes
Coenzyme Q

88. Features of electron carriers
All except coenzyme Q are proteins with prosthetic groups capable of being reversibly oxidized and reduced
Most are hydrophobic
Most of these intermediates occur in the membrane as large assemblies of proteins called respiratory complexes

89. Flavoproteins
Membrane-bound flavoproteins use either (FAD) or flavin adenine mononucleotide (FMN) as the prosthetic group
For example, NADH dehyrogenase and succinate dehydrogenase
Flavoproteins transfer both electrons and protons

90. Iron-Sulfur Proteins
Iron-sulfur proteins are also called nonheme iron proteins and have an iron-sulfur (Fe-S) center
The iron atoms in the center of the proteins are the actual electron carriers; these alternate between the Fe2+ (oxidized) and Fe3+ (reduced) states
They transfer one electron at a time, and no protons

91. Cytochromes
Cytochromes also contain iron, but as part of a porphyrin prosthetic group, heme
There are five types: b, c, c1, a, and a3
The iron atom of the heme group serves as the electron carrier and transfers one electron at a time and no protons

92. Figure 10-13

93. Copper-Containing Cytochromes
In addition to their iron atoms, cytochromes a and a3 contain a single copper atom bound to their heme group
It associates with the iron atom to form a bimetallic iron-copper (Fe-Cu) center
Copper ions can be reversibly converted from the oxidized (Cu2+) to the reduced form (Cu+) by accepting or donating single electrons

94. Copper-Containing Cytochromes (continued)
The iron-copper center plays a critical role in keeping an O2 molecule bound to the cytochrome oxidase complex
The oxygen is held there until it has picked up four electrons and four protons, at which point two water molecules are released

95. Coenzyme Q
The only nonprotein component of the ETS is coenzyme Q (CoQ), a quinone
Because of its ubiquitous occurrence in nature, it is also called ubiquinone
CoQ is reduced in two successive (one electron plus one proton) steps to semiquinone (CoQH) and then dihydroquinone (CoQH2)

96. Coenzyme Q (continued)
Unlike the proteins of the ETS, most of the CoQ is freely mobile in the inner mitochondrial membrane
They serve as a collection point for electrons from the reduced FMN and FAD-linked dehydrogenases in the membrane
A portion of the CoQ is tightly bound to specific respiratory complexes

97. Coenzyme Q (continued)
CoQ accepts both protons and electrons when it is reduced and releases both protons and electrons when it is oxidized
This is vital to its role in the active transport of protons across the inner mitochondrial membrane
It accepts protons on one side of the membrane, diffuses across and releases the protons

98. Figure 10-14

99. The Electron Carriers Function in a Sequence Determined by Their Reduction Potentials
The standard reduction potential (E0) for an electron carrier is a measure of its affinity for electrons.
For example, O2 accepting electrons is very favorable with E0 of V

100. The Electron Carriers Function in Sequence (continued)
NAD+ accepting electrons is favorable, too, but less so, with E0 of 0.32 V.

101. Understanding Standard Reduction Potentials
Arrange pairs of oxidized/reduced forms of the same molecule with the most negative reduction potential on the top and the rest in order of reduction potential
Under standard conditions, the reduced form of any redox pair can reduce the oxidized form of any redox pair below it in the list

102. Table 10-2

103. Energetics of redox reactions
The tendency of the reduced form of one pair to reduce the oxidized form of another pair can be quantified by determining the difference in Eo values between the two pairs

104. Energetics of redox reactions (continued)
For example, for transfer of electrons from NADH to O2

105. The relationship between DGo and DEo
DEo is a measure of thermodynamic spontaneity for the redox reaction between any two redox pairs under standard conditions
Where n = number of electrons transferred, F is the Faraday constant (23,062cal/mol*V)

106. Example: the reaction of NADH with oxygen
Two electrons are transferred, so we calculate DG0
The value is highly negative, so transfer of electrons this way is thermodynamically spontaneous

107. Ordering of the Electron Carriers
The position of each carrier is determined by its standard reduction potential
This means that electron transfer from NADH or FADH2 at the top to O2 at the bottom is spontaneous and exergonic

108. Most of the Carriers Are Organized into Four Large Respiratory Complexes
Although many electron carriers are part of the ETS, most are organized into multiprotein complexes
Most are thought to be organized into four different kinds of respiratory complexes

109. Figure 10-15

110. Properties of the Respiratory Complexes
Each respiratory complex consists of distinctive assembly of polypeptides and prosthetic groups
Complex I transfers electrons from NADH to CoQ and is called the NADH-coenzyme Q oxidation complex (or NADH dehydrogenase complex)

111. The respiratory complexes
Complex II transfers to CoQ the electrons derived from succinate in Reaction TCA-6 and it is called the succinate-coenzyme Q oxidoreductase complex, or succinate dehydrogenase
Complex III is called the coenzyme Q-cytochrome oxidoreductase complex because it accepts electrons from coenzyme Q and passes them to cytochrome c

112. The respiratory complexes (continued)
Complex III is also called cytochrome b/c1 complex
Complex IV transfers electrons from cytochrome c to oxygen and is called cytochrome c oxidase
For each pair of electrons transported through complexes I through IV, 10 protons are pumped from the matrix to the intermembrane space

113. Table 10-3

114. Figure 10-16A

115. Figure 10-16B

116. Figure 10-16C

117. Figure 10-16D

118. The Role of Cytochrome c Oxidase
Cytochrome c oxidase (complex IV) is the terminal oxidase, transferring electrons directly to oxygen
Cyanide and azide are toxic to most aerobic cells because they bind the Fe-Cu center of cytochrome c oxidase, blocking electron transport

119. Incomplete reduction of oxygen
Complexes I and III can also transfer electrons to oxygen, resulting in its incomplete reduction
This can generate toxic superoxide anion (O2) or hydrogen peroxide (H2O2), both of which contribute to cellular aging

120. The Respiratory Complexes Move Freely Within the Inner Membrane
The protein complexes of the mitochondrial inner membrane are mobile, free to diffuse within the membrane
Freeze-fracture analysis of membranes exposed to an electrical field demonstrate the free diffusion of the proteins
The inner membrane has no cholesterol and is very fluid, so the protein mobility is high

121. Respirasomes
Recent work suggests that the multiprotein respiratory complexes are organized into supercomplexes called respirasomes
The association of several of the TCA cycle dehydrogenases within the respirasomes suggests that they function to minimize diffusion distances

122. Coenzyme Q and cytochrome c
Coenzyme Q is the “funnel” that collects electrons from virtually every oxidation reaction in the cell
Coenzyme Q and cytochrome c are both small molecules that can diffuse rapidly within the membrane (coenzyme Q) or on its surface (cytochrome c)
They are both quite numerous, which accounts for observed rates of electron transfer

123. Electron Transport and ATP Synthesis Are Coupled Events
The crucial link between electron transport and ATP production is an electrochemical proton gradient
It is established by the directional pumping of protons across the membrane in which electron transport is occurring
ATP synthesis is coupled to electron transport

124. ATP synthesis is dependent on electron transport
Certain chemicals known as uncouplers can abolish the interdependence of the two processes
These allow continued electron transport and O2 consumption in the absence of ATP synthesis
Treatments that stop electron transport also inhibit ATP synthesis, so ATP synthesis is dependent on electron transport but the reverse is not true

125. Respiratory Control of Electron Transport
The availability of ADP regulates the rate of oxidative phosphorylation and thus of electron transport
This is called respiratory control
Electron transport and ATP generation will be favored when ADP concentration is high and inhibited when ADP concentration is low

126. The Chemiosmotic Model: The “Missing Link” Is a Proton Gradient
In 1961 Peter Mitchell proposed the chemiosmotic coupling model
The essential feature of the model is that the link between electron transport and ATP formation is the electrochemical potential across a membrane
The electrochemical potential is created by the pumping of protons across a membrane as electrons are transferred through the respiratory complexes

127. Coenzyme Oxidation Pumps Enough Protons to Form 3 ATP per NADH and 2 ATP per FADH2
The transfer of two electrons from NADH is accompanied by the pumping of a total of 10 protons (12 if the Q cycle is operating)
The number of protons required per molecule of ATP is thought to be 3 or 4, with 3 regarded as most likely
So, about 3 molecules of ATP are synthesized per NADH oxidized

128. Number of ATP generated is an estimate
FADH2 donates electrons to complex II with higher reduction potential, pumping 6 protons (8 if the Q cycle is operating)
So about 2 ATP are synthesized per FADH2
These values are estimates, affected by an organism’s specific ATP synthase and other factor

129. The Chemiosmotic Model Is Affirmed by an Impressive Array of Evidence
Since its initial formulation, the chemiosmotic model has become universally accepted as the link between electron transport and ATP synthesis
Several lines of evidence support the mode

130. 1. Electron Transport Causes Protons to Be Pumped Out of the Mitochondrial Matrix
Mitchell and Moyle demonstrated experimentally that the flow of electrons through the ETS is accompanied by the unidirectional pumping of protons across the inner mitochondrial membrane
One mechanism involves allosteric changes in protein conformation as electrons are transferred, allowing protons to be moved to the outer surface of the membrane

131. Figure 10-17

132. 2. Components of the Electron Transport System Are Asymmetrically Oriented Within the Inner Mitochondrial Membrane
Electron carriers of the ETS must be asymmetrically oriented in the membrane
Otherwise, protons would be pumped randomly in both directions
Labeling studies show that some parts of the respiratory complexes face inward, some are transmembrane proteins, others face outward

133. 3. Membrane Vesicles Containing Complexes I, III, or IV Establish Proton Gradients
Vesicles can be reconstituted from various components of the ETS
Vesicles that contain complex I, III, or IV are able to pump protons out of the vesicle, if provided with an appropriate oxidizable substrate

134. 4. Oxidative Phosphorylation Requires a Membrane-Enclosed Compartment
Without the mitochondrial membrane, the protein gradient that drives ATP synthesis could not be maintained
Electron transfer carried out by isolated complex cannot be coupled to ATP synthesis unless the complexes are incorporated into membranes that form enclosed vesicles

135. 5. Uncoupling Agents Abolish Both the Proton Gradient and ATP Synthesis
Dinitrophenol (DNP) is known to uncouple ATP synthesis from electron transport
When membranes are treated with DNP, they allow protons to cross the membrane freely, so that no proton gradient can be formed
ATP synthesis is abolished as well

136. 6. The Proton Gradient Has Enough Energy to Drive ATP Synthesis
The electrochemical proton gradient across the membrane involves both a membrane potential and a concentration gradient
A mitochondrion actively respiring has a membrane potential of about 0.16V (positive on the intermembrane space side) and a pH gradient of about 1.0 (higher on the matrix side)

137. Proton motive force
The electrochemical gradient exerts a proton motive force (pmf), that tends to drive protons back down their concentration gradient (back into the matrix)

138. Proton motive force in a mitochondrion
In a mitochondrion that is undergoing aerobic respiration, the pmf can be calculated:
Note that the membrane potential accounts for more than 70% of the pm

139. Pmf can be used to calculate DGo
The pmf can be used to calculate DGo as follows:
DG for phosphorylation of ADP is about 10–14 kcal/mol, so 3 or 4 protons would produce enough free energy to produce ATP

140. 7. Artificial Proton Gradients Are Able to Drive ATP Synthesis in the Absence of Electron Transport
Direct evidence for the model comes from work using vesicles or mitochondria that are exposed to artificial pH gradients
When the external proton concentration is increased, ATP is generated in response
So, ATP can be generated by a proton gradient, even when electron transport is not taking place

141. ATP Synthesis: Putting It All Together
Some of the energy of glucose is transferred to reduced coenzymes during glycolysis and the TCA cycle
This energy is used to generate an electrochemical proton gradient across the inner mitochondrial membrane
The pmf of that gradient is harnessed to make ATP

142. F1 Particles Have ATP Synthase Activity
Racker and colleagues took intact mitochondria and disrupted the membrane to form small vesicles called submitochondrial particles
These particles could carry out electron transport and ATP synthesis
By mechanical agitation or protease treatment, the F1 structures were isolated from the membranes

143. Figure 10-18A

144. Figure 10-18B

145. Figure 10-18C

146. Uncoupling synthesis of ATP and electron transport
F1 particles and membranous vesicles were separated by centrifugation
The membranes could still carry out electron transport, but could not synthesize ATP
The isolated F1 particles could synthesize ATP but could not carry out electron transport

147. Figure 10-18D .
147

148. Figure 10-18E .
148

149. Reconnecting synthesis of ATP and electron transport
Separated F1 particles and membranous vesicles were reconstituted
The reconstituted structures were capable of both electron transport and ATP synthesis
The F1 particles were called coupling factors and are now known to have ATP synthesizing activity

150. Figure 10-18E .
150

151. Figure 10-18F .
151

152. The FoF1 Complex: Proton Translocation Through Fo Drives ATP Synthesis by F1
The F1 complex is not directly membrane- bound, but is attached to the Fo complex that is embedded in the inner membrane
Fo acts as a proton translocator, the channel through which protons flow across the membrane
152

153. The FoF1 ATP synthase
Fo provides a channel for exergonic flow of protons across the membrane
F1 carries out the ATP synthesis, driven by the energy of the proton gradient
Together, they form a complete ATP synthase
153

154. Figure 10-19

155. Figure 10-19A

156. Figure 10-19B

157. Figure 10-19C

158. Figure 10-19D

159. Video: Rotation of ATP Synthase

160. ATP Synthesis by FoF1 Involves Physical Rotation of the Gamma Subunit
Paul Boyer proposed the binding change model in 1979
He proposed that each of the three b subunits of the F1 complex progresses through three different conformations
Each conformation has distinct affinities for the substrates, ADP and Pi, and the product, ATP .
160

161. The three conformations
Boyer named the conformations
L (for loose), which binds ADP and Pi loosely
T (for tight), which binds ADP and Pi tightly and catalyzes the formation of ATP
O (for open), with little affinity for either substrates or product .
161

162. Rotation of a and b with respect to g
Boyer proposed that at any time, each of the active sites is in a different conformation and the hexagonal ring of a and b subunits rotates relative to the central stalk containing the g subunit
The rotation was thought to be driven by the flow of protons through Fo
It is now known that it is the g subunit that actually rotates

163. The Binding Change Model in Action
Each b subunit passes through the O, L, and T conformations as the g subunit rotates 360 degrees
In Fo, the 10 c subunits each have an asp residue with an ionic bond to an arg residue on the immobile a subunit
When taken up, a proton neutralizes the asp, disrupting the ionic bond, and rotating the c10 ring (and attached g subunit) 1/10th turn

164. The binding change model (continued)
As the ring turns, the asp in the adjacent residue loses a proton and forms an ionic bond to arg in the a subunit
As 10 protons pass through the membrane via the a subunit, the ring goes through one complete rotation

165. Steps of the model
Step 1a: The b1 subunit in the O conformation shifts to the L conformation (loosely binding ADP and Pi) when the flow of protons through Fo causes a 120-degree rotation of the g subunit
Step 1b: ATP is synthesized by b3
Step 2a: The g subunit rotates another 120 degrees, inducing b1 to shift to the T conformation

166. Steps of the model (continued)
Step 2b: The b1 subunit has an increased affinity for ADP and Pi, allowing spontaneous formation of ATP
Step 3a: The g subunit rotates another 120 degrees, inducing b1 to shift to the O conformation, releasing ATP
Following ATP generation by the b2 unit (step 3b), the cycle is complete

167. Figure 10-20
167

168. Video: ATP Synthase 3-D Structure

169. Spontaneous Synthesis of ATP?
Each of the three steps at which ATP is formed occurs without a direct input of energy
However, ATP synthesis is highly endergonic (in dilute aqueous solution)
The catalytic site of a b subunit is such that the charge repulsion between ADP and Pi is minimized, favoring ATP formation with a DGo close to zero

170. Synthesis of ATP without thermodynamic cost?
ATP synthesis does not proceed without thermodynamic cost
The needed input of energy occurs elsewhere in the cycle
Energy comes from the proton gradient generated by electron transport and transmitted through rotation of the g subunit

171. Synthesis of ATP without thermodynamic cost?
ATP synthesis does not proceed without thermodynamic cost
The needed input of energy occurs elsewhere in the cycle
Energy comes from the proton gradient generated by electron transport and transmitted through rotation of the g subunit

172. Figure 10-21

173. The Chemiosmotic Model Involves Dynamic Transmembrane Proton Traffic
There is continuous, dynamic two-way proton traffic across the inner membrane
NADH sends 10 protons across via complexes I, III, and IV; FADH2 sends 6 across, via complexes II, III, and IV
Assuming that 3 protons must return through FoF1 per ATP generated, this means 3 ATP per NADH and 2 per FADH2 are generated
173

174. Figure 10-22

175. Aerobic Respiration: Summing It All Up
As carbohydrates and fats are oxidized to generate energy, coenzymes are reduced
These reduced coenzymes represent a storage form of the energy released during oxidation
This energy can be used to drive ATP synthesis as the enzymes are reoxidized by the ETS
175

176. Summing it all up (continued)
As electrons are transported from NADH or FADH2 to O2, they pass through respiratory complexes where proton pumping is coupled to electron transport
The resulting electrochemical gradient exerts a pmf that serves as the driving force for ATP synthesis
176

177. The Maximum Yield of Aerobic Respiration Is 38 ATPs per glucose
The maximum ATP yield per glucose under aerobic conditions:
Including the summary reactions of glycolysis and the TCA cycle to this gives:
177

178. 1. Why Does the Maximum ATP Yield in Eukaryotic Cells Vary Between 36 and 38 ATPs Per Glucose?
Glycolysis produces two NADH per glucose in the cytosol, and catabolism of pyruvate produces eight more in the mitochondrial matrix
NADH in the cytosol cannot enter the matrix to deliver its electrons to complex I
Instead the electrons and H+ ions are passed inward by an electron shuttle system
178

179. Electron shuttle systems
There are several electron shuttle systems that differ in the number of ATP molecules formed per NADH molecule oxidized
Electron shuttle systems consist of one or more electron carriers that can be reversibly reduced, with transport proteins in the membrane for both reduced and oxidized forms of carrier
179

180. The glycerol phosphate shuttle
The glycerol phosphate shuttle is one type of electron shuttle
NADH in the cytosol reduces dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, which is transported into the mitochondrion
There it is reoxidized to DHAP using FAD, so the electrons bypass complex I, generating 2 ATP instead of 3
180

181. Figure 10-23

182. 2. Why Is the ATP Yield of Aerobic Respiration Referred to as the “Maximum Theoretical ATP Yield”?
Yields of 36 to 38 ATP per glucose are possible, only if the energy of the electrochemical gradient is only used for ATP synthesis
This is not realistic, because the pmf of the proton gradient is used for other processes too, as needed by the cell

183. Figure 10-24

184. Figure 10-24A

185. Figure 10-24B

186. Figure 10-24C

187. Figure 10-24D

188. Figure 10-24E

189. Aerobic Respiration Is a Highly Efficient Process
To determine efficiency of respiration, we need to determine how much of the energy of glucose is preserved in the resulting 36–38 ATP
DGo for glucose  CO2 + H2O is 686 kcal/mol
ATP hydrolysis under cellular conditions is about 10 to 14 kcal/mol

190. Efficiency of aerobic respiration
For 36–38 ATP, assuming a value of 10 kcal/mol, the energy per mole of glucose is about 360–380 kcal conserved
This efficiency of 52–55% is well above that obtainable from the most efficient machines created