© 2012 Pearson Education, Inc. CHAPTER 10 Cellular Respiration

© 2012 Pearson Education, Inc.

CHAPTER 10Cellular Respiration


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


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


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


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


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


Figure 10-1


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


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


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)


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


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


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


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


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


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


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


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


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


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


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


Table 10-1


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


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)


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


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


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


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


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


Figure 10-6


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)


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)


Conversion of pyruvate

• The conversion of pyruvate to acetyl CoA can be summarized as follows:

–


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


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


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


TCA-3, TCA-4

• Isocitrate is oxidized by isocitrate dehydrogenase to oxalosuccinate, with NAD+ as the electron acceptor

• Oxalosuccinate immediately undergoes decarboxylation to form -ketoglutarate (5C)(TCA-3)

• -ketoglutarate is oxidized to succinyl CoA, by -ketoglutarate dehydrogenase (TCA-4)


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)


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


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)


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


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


Summing up the TCA cycle

• The TCA cycle can be summarized as follows:

• Including glycolysis, pyruvate decarboxylation, and the TCA cycle


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


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


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)


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


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


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 -oxidation


-oxidation

• -oxidation is a catabolic process that generates acetyl CoA and the reduced coenzymes NADH and FADH2

• -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


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


Steps of-oxidation

• Each cycle involves

– 1.Oxidation

– 2. Hydration

– 3. Reoxidation

– 4. Thiolysis

• The result is the production of one FADH2, one NADH, and one acetyl CoA per cycle


Formation of fatty acyl CoA

• -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


Degradation of fatty acyl CoA

• An integral membrane dehydrogenase oxidizes the fatty acyl CoA, forming a double bond between the and 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)


Degradation of fatty acyl CoA (continued)

• Another dehydrogenase oxidizes the -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 and carbons is broken by a thiolase and a two-carbon fragment is transferred to a second acetyl CoA (FA-5)


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


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


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


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


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


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


The TCA cycle has a central role in some anabolic processes

• -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


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


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


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


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

•

•


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)


The Electron Transport System Consists of Five Kinds of Carriers

• Flavoproteins

• Iron-sulfur proteins

• Cytochromes

• Copper-containing cytochromes

• Coenzyme Q


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


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


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


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


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


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


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)


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


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


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 +0.816 V

•


The Electron Carriers Function in Sequence (continued)

• NAD+ accepting electrons is favorable, too, but less so, with E0 of 0.32 V.

•


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


Table 10-2


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 Eo values between the two pairs


Energetics of redox reactions (continued)

• For example, for transfer of electrons from NADH to O2


The relationship between Go and Eo

• Eo 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)


Example: the reaction of NADH with oxygen

• Two electrons are transferred, so we calculate G0

• The value is highly negative, so transfer of electrons this way is thermodynamically spontaneous


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


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


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)


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


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


Table 10-3


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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)


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)

•


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


Pmf can be used to calculate Go

• The pmf can be used to calculate Go as follows:

• G for phosphorylation of ADP is about 10–14 kcal/mol, so 3 or 4 protons would produce enough free energy to produce ATP


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


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


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


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


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


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


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


Figure 10-19


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 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


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


Rotation of and with respect to

• Boyer proposed that at any time, each of the active sites is in a different conformation and the hexagonal ring of and subunits rotates relative to the central stalk containing the subunit

• The rotation was thought to be driven by the flow of protons through Fo

• It is now known that it is the subunit that actually rotates


The Binding Change Model in Action

• Each subunit passes through the O, L, and T conformations as the 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 subunit) 1/10th turn


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


Steps of the model

• Step 1a: The 1 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 subunit

• Step 1b: ATP is synthesized by 3

• Step 2a: The subunit rotates another 120 degrees, inducing 1 to shift to the T conformation


Steps of the model (continued)

• Step 2b: The 1 subunit has an increased affinity for ADP and Pi, allowing spontaneous formation of ATP

• Step 3a: The subunit rotates another 120 degrees, inducing 1 to shift to the O conformation, releasing ATP

• Following ATP generation by the 2 unit (step 3b), the cycle is complete


Figure 10-20


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 subunit is such that the charge repulsion between ADP and Pi is minimized, favoring ATP formation with a Go close to zero


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 subunit


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 subunit


Figure 10-21


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


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


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


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:


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


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


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


Figure 10-23


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


Figure 10-24


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

• Go for glucose CO2 + H2O is 686 kcal/mol

• ATP hydrolysis under cellular conditions is about 10 to 14 kcal/mol


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

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© 2012 Pearson Education, Inc. CHAPTER 10 Cellular Respiration