I. Overview of Aerobic Metabolism.

Aerobic (oxygen-using) metabolism extracts energy from carbohydrate sources, fatty acids and amino acids. While glycolysis yields two moles of ATP from one mole of glucose, full oxidation of glucose by aerobic respiration produces ~30 moles of ATP.

Aerobic metabolism occurs in three phases. First, carbohydrates are oxidized to CO2, producing the energy-rich molecules NADH and FADH2. Electrons from NADH and FADH2 are then passed along the electron transport chain to the terminal electron acceptor O2. The free energy released in electron transport is captured by coupling it to the export of protons across the mitochondrial inner membrane. Finally, the free energy of the electrochemical proton gradient is used to synthesize ATP from ADP, Pi and H+, and to export ATP from the mitochondria.

Many catabolic pathways (carbohydrate, amino acid, fatty acid, and ketone body) converge at the TCA cycle. Many anabolic pathways depart from the TCA cycle, including synthesis of fatty acids, amino acids, purine bases, heme, cholesterol, steroid hormones and ketone bodies. The TCA cycle provides carbon skeletons in a variety of different oxidation states for these biochemical processes.










Four Coupled Reactions Constitute Aerobic Metabolism:

A. Conversion of Pyruvate to Acetyl CoA.

Pyruvate + CoA + NAD+ ---> Acetyl CoA + CO2 + NADH

B. The Tri-Carboxylic Acid (TCA) Cycle [Also called the Krebs Cycle and the Citric Acid Cycle].

Acetyl CoA + 2 H2O + 3 NAD+ + FAD + GDP + Pi --->

2 CO2 + CoA + 3 NADH + FADH2 + GTP + 2 H+

C. Electron Transport.

1/2 O2 + H+ + NADH + 10 ---> H2O + NAD+ + 10


1/2 O2 + FADH2 + 6 ---> H2O + FAD + 6

D. ATP Synthesis.

3 + ADP + Pi + H+ ---> 3 + ATP + H2O


Aerobic Metabolism in Eukaryotes Occurs in Mitochondria. Mitochondria are oval organelles ~2 mM long and ~0.5 mM in diameter. They are present in most differentiated tissues, with the exception of mature erythrocytes, cornea and lens tissue. The outer membrane is permeable to metabolites because it contains many copies of the porin protein. The inner membrane is impermeable to polar metabolites. Specific transporters are utilized to pass small molecules across it.

The inner membrane contains the electron transport proteins and the F0F1 ATP synthase. Matrix proteins include pyruvate dehydrogenase, the TCA cycle enzymes, the enzymes of fatty acid catabolism, and the enzymes that catalyze the first steps of the urea cycle. The highly folded nature of the mitochondrial inner membrane results in a high ratio of membrane surface area to matrix volume, which allows rapid exchange of NADH between the soluble enzymes of the TCA cycle and the membrane bound electron transport machinery.

Protons are pumped out of the mitochondrial matrix and into the cytosol, producing a pH gradient of 1.5 pH units and a 140 millivolt electrostatic potential difference across the inner membrane. Mitochondria act as intracellular chemical reactors, and as chemical/electrical capacitors.



A. Oxidative Decarboxylation of Pyruvate: Chemistry. The reaction catalyzed by the pyruvate dehydrogenase complex occurs in three steps, catalyzed by three different subunits of a multi-protein complex in the mitochondrial matrix.

1. Decarboxylation of an a-keto acid. The mechanism utilizes the catalytic cofactor thiamine pyrophosphate (TPP) to stabilize an acyl carbanion. TPP acts as an electron sink. The same mechanism is used by the enzyme a-ketoglutarate dehydrogenase (TCA cycle) and transketolase (pentose phosphate pathway). The acyl carbanion is oxidized to a thiolester by the intramolecular disulfide of lipoic acid. The thiolester represents a high energy bond (cf. the acyl enzyme intermediate of glyceraldehyde-3-phosphate dehydrogenase). Decarboxylation helps to drive the reaction forward. Both steps are catalyzed by the pyruvate decarboxylase or E1 subunit of the complex.





















2. The acyl group is transferred onto the thiol of the catalytic cofactor A (CoA). Catalyzed by the transacetylase or E2 subunit of the complex.










3. Reduced lipoic acid is reoxidized by transferring electrons to nicotine-adenine dinucleotide (NAD+) through a bound flavin-adenine dinucleotide (FAD) intermediate. Catalyzed by the dihydrolipoyl dehydrogenase or E3 subunit of the complex.




















B. Reactions of the TCA cycle: Chemistry. The TCA cycle oxidizes a two-carbon (C2) unit completely to CO2. The reactions recapitulate similar transformations that occur during glycolysis. In summary, a C2 unit from acetyl CoA is attached to the C4 "carrier" oxaloacetate to form the C6 molecule citrate. Citrate is oxidatively decarboxylated twice to from the reduced C4 unit succinate. Succinate is then oxidized to regenerate the C4 "carrier" oxaloacetate.





Mechanistic Notes

1. Proteins often produce chiral products from achiral starting materials, for example the production of l-malate from fumarate.
















2. Achiral symmetric starting materials can react asymmetrically if they are prochiral. For example, the acetyl CoA C2 unit added to oxaloacetate is not the one oxidized to CO2. Citrate is prochiral, fumarate is not.
















(a) A molecule is optically inactive (achiral) if it can be superimposed on it’s mirror image.

(b) Two substituents are indistinguishable if a rotation exists which maps one substituent into the other, but leaves the rest of the structure chemically invariant.

(c) Achiral molecules with distinguishable substituents are said to be prochiral.



















The nicotinamide ring of NADH is prochiral, and reacts asymmetrically.


















3. The TCA cycle feeds a number of anabolic pathways.
























4. "Fat burns in the flame of carbohydrate". Oxaloacetate is not created by the TCA cycle, but is required in a catalytic role for the cycle to function. In vertebrates, oxaloacetate can not be synthesized from acetyl CoA, the primary breakdown product of fat fuels. As C4, C5 and C6 intermediates are siphoned off of the TCA cycle, new oxaloacetate must be provided. Oxaloacetate can be generated from pyruvate as described below.


Anaplerotic Synthesis of Oxaloacetate from Pyruvate

Pyruvate can be converted to lactate by anaerobic fermentation, or can enter the TCA cycle through pyruvate dehydrogenase. A third fate of pyruvate is conversion to oxaloacetate by pyruvate carboxylase. Pyruvate carboxylase requires the catalytic cofactor biotin. The CO2~biotin bond is formed from bicarbonate and biotin at the expense of one ATP molecule, and has a high CO2 group-transfer potential (cf. creatine phosphate). Pyruvate carboxylase is allosterically activated by high concentrations of acetyl CoA, which signal the need for more oxaloacetate to feed the TCA cycle. Pyruvate carboxylase also catalyzes the first step of gluconeogenesis, which will be discussed later.



















Regulation of Pyruvate Dehydrogenase

In vertebrates, the formation of acetyl CoA from pyruvate is irreversible; acetyl CoA can’t be recycled into C3 or larger units. Consequently, pyruvate dehydrogenase represents a critical decision point in metabolism. It is tightly regulated by phosphorylation/dephosphorylation of the E1 pyruvate decarboxylase subunit.

Inactivation of Pyruvate Dehydrogenase (phosphorylation):

=>Kinase Stimulation: High NADH/NAD+, acetyl CoA/CoA or ATP/ADP ratios.

Activation of Pyruvate Dehydrogenase (dephosphorylation):

=>Kinase Inactivation: Pyruvate

=>Phosphatase Stimulation: Ca2+ (vasopressin, a-adrenergic agonists), insulin


Regulation of the TCA Cycle

The three steps of the TCA cycle characterized by large negative free energies (citrate synthase, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase) are regulated allosterically by ATP and ADP levels.


























O2 and the energy charge of the cell directly regulate the TCA cycle by effecting substrate availability. Within mitochondria, the level of acetyl CoA usually exceeds the NAD+ equivalents available for the oxidative steps of the TCA cycle. In actively respiring cells, NAD+ is constantly regenerated by oxidative phosphorylation and ATP consumption. Interruption of oxidative phosphorylation or ATP consumption rapidly leads to the reduction of all of the available NAD+ (as well as the electron acceptors of the electron transport chain), bringing the TCA cycle to a grinding halt. Thus (a) availability of O2 (the terminal electron acceptor in oxidative phosphorylation) and (b) availability of ADP and Pi (which reflect the energy needs of the cell) directly regulate the TCA cycle by effecting the NAD+/NADH ratio inside mitochondria.


Clinical Correlations

C. Electron Transport

Oxidative phosphorylation consists of two mechanistic steps: (a) Electron transport: the transfer of electrons from NADH and FADH2 to O2 through a series of intermediate electron carriers, with concomitant export of protons from mitochondria and (b) ATP synthesis: utilization of the electrochemical potential energy of the proton gradient to synthesize ATP from ADP+Pi+H+. We begin with electron transport.

1/2 O2 + H+ + NADH + 10 ---> H2O + NAD+ + 10


1/2 O2 + FADH2 + 6 ---> H2O + FAD + 6














Where are the Electrons?

Recognizing exchange of electrons by oxidative/reductive organic transformations can be difficult. A simple procedure (Introduction to Organic Chemistry, 3rd Ed., A Streitwieser Jr. and C. Heathcock, Macmillan Publishing Co., N.Y., 1985 pg. 213) can clarify matters:

(1) Write the starting material and the product of the transformation.

(2) Balance the equation for hydrogen atoms by adding protons (H+).

(3) Balance the equation for charge by adding electrons (e-).





















Energy from Electron Transport

Pairs of compounds that can be interconverted by addition/removal of electrons and protons are called redox couples. The species with bound electrons is said to be reduced; the species that has given up electrons is said to be oxidized. Different redox couples have different electron affinities. The electron affinity of a redox couple is determined electrochemically by measuring its reduction potential, which has units of volts. The H+/H2 couple in aqueous solution at pH 7 and 1 atmosphere of H2 is used as the reference half cell, and is defined to have a reduction potential of 0 volts. Redox couples with low electron affinities have negative reduction potentials; redox couples with high electron affinity have positive reduction potentials.






























Redox couples with low electron affinity (for example NADH/NAD+) are poised to donate electrons to electron-greedy redox couples (for example O2/H2O). This electron transport occurs with a net negative free energy. DG for the transport reaction can be calculated from the reduction potentials of the donor and acceptor pairs using the Nernst equation. As for all chemical reactions, DG depends on the concentrations of the starting materials and the products.


DG = -nF{(acceptor) - (donor)} + RT*ln


n is the number of electrons transferred in the process and F is a proportionality constant called the Faraday, which converts energy in units of volts (ergs/esu/300) to energy in units of kcal/mol; F has a value of 23.06 kcal/molV-1. [Reactant] and [product] are expressed in units of standard state concentration (1M for all species except H2O, OH- and H+, which have standard state concentrations of 55 M, 10-7 M and 10-7 M respectively; the standard state for gases is 1 atmosphere pressure above the solution). Consider the transfer of two electrons from NADH/NAD+ to oxygen under standard state conditions:

Acceptor: 1/2 O2 + 2 H+ + 2 e- ---> H2O = +0.82 V

Donor: NAD+ + H+ + 2 e- ---> NADH = -0.32 V

Total: 1/2 O2 + NADH + H+ ---> H2O + NAD+

(acceptor) - (donor) = +1.14 V


DG0’ = -nF{+1.14 V} + RT*ln{1} = -2*26.03*1.14 = -52.6 kcal/mol

Under standard state conditions, transfer of one pair of electrons from NADH to O2 releases -53.6 kcal/mol! The free energy of the corresponding reaction in mitochondria depends on (a) the NADH/NAD+ ratio (b) the H+ concentration and (c) the O2 concentration.


Biological Electron Carriers

Five major classes of electron carriers (redox couples) participate in aerobic metabolism. We have already encountered three: NADH/NAD+, FADH2/FAD, and the disulfide bonded cofactor lipoic acid. Two additional electron carriers essential to aerobic metabolism are (a) ubiquinone (QH2/Q) and (b) the transition metals iron and copper. Iron is found in iron-sulfur complexes (Fe-S) and also as heme-bound iron, and cycles between ferric (Fe3+) and ferrous (Fe2+) forms. The electron carriers of aerobic metabolism differ from each other in reduction potential, electron capacity, and mechanism of reduction/oxidation (two-electron hydride transfer versus one-electron transfer).

NADH/NAD+: Two electron capacity, hydride transfer.

FADH2/FAD: Two electron capacity, one electron transfer.

Lipoic Acid: Two electron capacity, one electron transfer.

QH2/Q: Two electron capacity, one electron transfer.

Fe-S, heme-bound metal: One electron capacity, one electron transfer.























Electron Transport Chain and Proton Pumping

The transfer of electrons from NADH to O2 releases substantial free energy. This energy is harvested by coupling electron transport to the export of protons out of the mitochondrial matrix, against an electrochemical gradient. To capture the energy most efficiently, electrons are passed stepwise through a series of ~10 electron carriers with reduction potentials intermediate between those of NADH and O2. The electron carriers of the electron transport chain are organized into four multiprotein complexes. These complexes are connected by two mobile electron carriers, ubiquinone and the Fe-heme containing protein cytochrome c. Ubiquinone diffuses freely in the hydrocarbon core of the inner mitochondrial membrane. Cytochrome c (Cyt c) diffuses freely over the outer surface of the inner mitochondrial membrane.



























1. Complex I, NADH dehydrogenase, 880 kD, _34 subunits.

NADH + H+ + Q + 4 ---> NAD+ + QH2 + 4 D = 0.35 V

The free energy resulting from transfer of electrons from NADH to the mobile carrier ubiquinone is used to pump four protons out of the mitochondrial matrix.

2. Complex II, succinate dehydrogenase (TCA cycle), 140 kD, 4 subunits.

Succinate + Q ---> Fumarate + QH2 D = 0.01 V

Electrons released by the oxidation of succinate to fumarate feed into the electron transport chain at the level of QH2, below NADH. No protons are pumped in this reaction, because the free energy of the electron transfer is close to zero.

3. Complex III, cytochrome reducatase, 250 kD, 10 subunits.

QH2 + 2 Cyt c(Fe3+) + 2 ---> Q + 2 Cyt c(Fe2+) + 2 D = 0.21 V

The free energy released by transfer of electrons from QH2 to Cyt c is used to pump two protons out of the mitochondrial matrix. Complex III uses a recycling mechanism to connect a two electron donor (QH2) to a one electron recipient (Cyt c ) as illustrated below.















4. Complex IV, cytochrome oxidase, 160 kD, 10 subunits.

4 Cyt c(Fe2+) + 4 H+ + O2 + 4 ---> 4 Cyt c(Fe3+) + 2 H2O + 4


D = 0.57 V

In the final step of the electron transport chain, 4 electrons from the one electron donor Cyt c are used to reduce oxygen to water, with concomitant export of four protons from the mitochondrial matrix. Complex IV solves a challenging mechanistic problem: to reduce completely oxygen without releasing the toxic intermediates superoxide anion, O2-, and hydrogen peroxide, HOOH. A tandem pair of metals, Fe-heme a3 and CuB, carry out the reaction as illustrated below.




















Phagocytic white blood cells of the mammalian immune system deliberately produce superoxide anion and peroxide in order to destroy encapsulated pathogens. However, most cells produce the proteins superoxide dismutase and catalase to police against these toxic species. Superoxide dismutase catalyzes the conversion superoxide to peroxide and oxygen while catalase catalyzed the conversion of peroxide to water and oxygen.

superoxide dismutase: 2 O2- + 2 H+ ---> O2 + H2O2

catalase: 2 H2O2 ---> 2 H2O + O2


How is Electron Transport Coupled to Proton Pumping?

The mechanism that couples electron transport to proton pumping is not well understood. Based on studies of a bacterial proton pump called bacteriorhodobsin, the following model has been proposed.

Model: The pump cycles between an inward conformation and an outward conformation depending on whether a bound prosthetic group is reduced or oxidized. The pump contains a protonatable side-chain R with pKA > 8.5 in the inward state and pKA < 7 in the outward state. Finally, R can only exchange protons externally in the outward state and only internally in the inward state. Suppose that the mitochondrial matrix has a pH of 8.5 and the cytosol has a pH of 7. R will protonate while facing inward and deprotonate while facing outward, effectively pumping one proton out of the matrix for each reduction-oxidation cycle.

















(Note: To achieve tight coupling between proton pumping and electron transport, two conditions must be met. First, the prosthetic group must accept electrons only when the pump faces inward, and only donate electrons when the pump faces outwards. Second, the reduction of the bound prosthetic group must be kinetically slow when R is deprotonated. Such kinetic gating could result if the charge on RH+ helped to stabilize the transition state for the reduction reaction, for example).


Energy Stored in the Proton Gradient

As a consequence of proton export, the pH of the mitochondrial matrix in actively respiring tissue is 1.4 units higher than the pH of the cytosol. In addition, the net excess of positive charges in the cytosol (and net excess of negative charges in the matrix) produces a 140 millivolt electric potential drop across the mitochondrial inner membrane. Both the chemical gradient (DpH) and the electric potential difference (milivolts) contribute to the release of free energy when protons reenter the mitochondrial matrix.

To calculate the chemical gradient contribution, consider the reaction  ---> . In the standard state with 1 M H+ on both sides of the membrane, the free energy of the reaction is zero, i.e. DG0’=0. However, when a concentration gradient of protons exists:


DGchemical = DG0’ + RT*ln = {pHout-pHin}*RT*ln{10} = 1.9 kcal/mol

for a pH difference of 1.4 pH units at 25° C.

To calculate the electric potential contribution, we multiply the potential difference in volts by F, the free energy released when one mole of electrons passes through an electric potential of 1 volt.


DGelectrical = F*Volts = 23.06*0.14 = 3.2 kcal/mol

The chemical and electric DG’s sum to 5.1 kcal/mol, the free energy for reentry of protons into the mitochondrial matrix. As electrons from one mole of NADH pass along the electron transport chain to oxygen, ten moles of mitochondrial protons are exported. The ten moles of exported protons represent a captured free energy of 10*5.1 = 51 kcal/mol!


Inhibitors of Electron Transport and Artificial Electron Acceptors

The insecticide rotenone, the barbiturate drug amytal, the antibiotic antimycin and the poisons cyanide, azide and carbon monoxide inhibit electron transport by blocking specific steps of the transport chain. These inhibitors were used to establish the order of electron carriers in the chain. Carriers upstream of an inhibitor become fully reduced as electrons flow in above the block, while carriers downstream become fully oxidized as electrons flow out below the block (the oxidation state of many carriers in the electron transport chain can be determined spectroscopically). Addition of artificial electron acceptors of variable to cyanide-blocked mitochondria has also been used to measure the effective reduction potential of intermediates along the transport chain.












D. ATP Synthesis

The last step of aerobic metabolism is the conversion of energy from electron transport into the high-energy phosphoryl bond of ATP. Early theories for the mechanism of ATP synthesis proposed substrate-level phosphorylation of ADP by electron transport intermediates with high Pi group transfer potentials (e.g. formation of ATP from 1,3 bisphosphoglycerate in glycolysis). A competing theory proposed that the free energy of oxidation is trapped in activated conformations of the electron transport proteins, which subsequently drive ATP synthesis. The correct explanation was put forward by Peter Mitchell in 1961. Mitchell’s chemiosmotic hypothesis proposed that the events of electron transport pump protons out of mitochondria (as we have seen), and that the resulting electrochemical proton gradient is used to synthesize ATP from ADP + Pi + H+. Subsequently, a protein called the F0F1 ATP synthase was purified from mitochondria and shown to catalyze the following reaction:

3 + ADP + Pi + H+ ---> 3 + ATP + H2O

The F0F1 ATP synthase has the shape of a lollipop. The F1 domain forms the head of the lollipop, faces towards the inside of the mitochondrial matrix, and carries out ATP synthesis. The F0 domain forms the stem of the lollipop, resides in the inner mitochondrial membrane, and is a proton channel. Both domains have trimer symmetry. The F0F1 ATP synthase lollipops appear as a dense array of studs on the inner mitochondrial membrane when examined under the electron microscope.





























A key experiment performed by W. Stoeckenius and E. Racker demonstrates that the electron transport chain and the F0F1 ATP synthase are biochemically independent systems, coupled only by the proton motive force across the mitochondrial inner membrane. Stoeckenius and Racker reconstituted vesicles containing purified F0F1 ATP synthase and bacteriorhodopsin, a bacterial proton pump that responds to light. When illuminated, the bacteriorhodobsin pump created a proton gradient across the vesicle membrane, and powered ATP synthesis by the F0F1 ATP synthase. Thus the entire respiratory chain could be replaced by an unrelated proton pumping apparatus.



















Structural Biochemistry of the F0F1 ATP Synthase

The F1 domain of the ATP synthase consists of five polypeptides (a-e) with an a3b3gde stoichiometry. In the 2.8 Å X-ray crystal structure of the F1 subunit reported in 1994, the three a and three b subunits (which are 20% identical in sequence) are arranged with pseudo-six fold symmetry like the segments of an orange. The three ab dimers that make up the orange are chemically identical, but they adopt three different structural conformations, designated the L (loose), T (tight) and O (open) conformations. The b subunit in the L conformation is observed to bind to ADP, the b subunit in the T conformation is observed to bind to an ATP analogue, and the b subunit in the O conformation is observed to have an empty and disordered nucleotide binding site. The conformational asymmetry between the three b subunits appears to result from interactions with the g polypeptide. The g polypeptide forms a curved, antiparallel coiled coil which makes distinct interactions with each of the b subunits. The g polypeptide faces into the stalk connecting the F0 and F1 domains of the ATP synthase.





















Enzyme Bound ATP Forms Without Energy Input

A key insight into the mechanism of the F0F1 ATP synthase is provided by isotope exchange experiments. When purified F1 subunit is incubated in H218O with ADP and Pi (no proton gradient), 18O is rapidly incorporated in inorganic phosphate. The rate of incorporation suggests that enzyme-bound {ADP + Pi + H+} and enzyme-bound {ATP + H2O} have similar free energies. This is an example of substrate binding energy altering a chemical equilibrium on an enzyme, and shows that energy from the proton gradient is not used to form ATP, but to release it from the synthase.


















The Binding-Change Mechanism

The biochemical and structural experiments discussed above support the binding-change mechanism for ATP synthesis proposed by H. Boyer. The key assumptions of the model are outlined below.

1. At any instant in time, the three nucleotide binding sites of the F1 subunit are in different states; one adopts the L conformation, one the T conformation and one the O conformation.

2. Rotation of the g polypeptide by 120° increments causes each nucleotide binding site to change state in the sequence L -> T -> O -> L. (This has been observed under the microscope!).

3. The g polypeptide transduces energy from the F0 domain to the F1 domain. Rotation of the g polypeptide by 120° is coupled to, and energetically driven by, the import of 3 H+ through the F0 proton channel. Rotation of the g polypeptide by a proton motive force may be mechanistically related to rotation of the bacterial flagellar motor (see Stryer, pg. 412).

4. Nucleotide binding free energies in the L, T and O states at physiologic nucleotide, proton and phosphate concentrations are roughly as follows:

Binding Free Energy

Conformation {ADP + Pi + H+} {ATP + H2O}

L state Negative Positive

T state Negative Negative

O state Positive Positive

The change in binding affinity of the b subunits for {ADP + Pi + H+} and {ATP + H2O} as the g polypeptide rotates results in net ATP synthesis (see illustration below). Thus the electochemical energy of the proton gradient is converted into a high-energy phosphoryl bond.

















Transport of NADH and ATP Across the Inner Mitochondrial Membrane

Finally, ATP produced by the F0F1 ATP synthase must be delivered from the mitochondrial matrix to the cytosol, where it can power cellular processes. However, charged metabolites do not cross the mitochondrial inner membrane by passive diffusion. A family of antiporter proteins exist that exchange one metabolite on the matrix side of the membrane for another metabolite on the cytosolic side. Many of these transporters are directional (the forward and reverse transport reactions are not of equal free energy), and are driven by the chemical or electric potential of the proton gradient.

The adenine nucleotide transporter exchanges ATP4- in the matrix for ADP3- in the cytosol. Transport is driven by the 140 millivolt electric potential across the membrane, as the exchange results in a unit negative charge passing out of the mitochondria. The phosphate transporter exchanges cytosolic H2PO4- for matrix OH- in an electroneutral process. Phosphate transport is driven by the chemical potential of the proton gradient.

No transporter exists to deliver the NADH produced during glycolysis to the mitochondrial matrix. Instead, two indirect mechanisms ferry electrons from cytosolic NADH to the electron transport chain.

In the malate-aspartate shuttle, cytosolic oxaloacetate is reduced to malate by cytosolic NADH (this is the final reaction of the TCA cycle run in reverse). Malate is transported into mitochondria in exchange for mitosolic a-ketoglutarate by the malate-a-ketoglutarate transporter. The malate is reoxidized to oxaloacetate, regenerating one equivalent of NADH inside the mitochondrial matrix. To complete the cycle, oxaloacetate must be returned to the cytosol. Oxaloacetate is first transaminated to aspartate by aspartate aminotransferase, a pyridoxal phosphate requiring enzyme that will reappear in the lectures on amino acid metabolism. The aspartate-glutamate transporter delivers aspartate to the cytosol, where the reverse transamination reaction regenerates oxaloacetate. The balanced reaction is:

2 + 2 ---> 2 + 2 .

In the glycerol phosphate shuttle, cytosolic NADH is used by glycerol 3-phosphate dehydrogenase to reduce dihydroxyacetone phosphate to glycerol 3-phosphate. The glycerol 3-phosphate diffuses to the mitochondrial inner membrane, where a mitochondrial glycerol dehydrogenase with a bound FAD prosthetic group reoxidizes it to dihydroxyacetone phosphate. The bound FADH2 in turn reduces Q to QH2. The glycerol phosphate shuttle is less energetically efficient than the malate-asparatate shuttle because it introduces electrons into the electron transport chain at the level of Q.


Counting ATP

We are now in a position to calculate how many moles of ATP are generated by complete oxidation of glucose to CO2. First, conversion of glucose to two molecules of pyruvate by glycolysis yields two moles of ATP and one mole of NADH. Oxidation of pyruvate by pyruvate dehydrogenase and the TCA cycle yields two moles of GTP (convertible to ATP by nucleoside diphosphate kinase), eight moles of NADH, and two moles of QH2. Assuming transport by the glycerol phosphate shuttle, the two moles of NADH produced by glycolysis are converted to two moles of QH2. Thus four moles of ATP, eight moles of NADH and four moles of QH2 are produced by complete oxidation of one mole of glucose.

For each mole of ATP + H2O produced from ADP + Pi + H+, four moles of are consumed, three by the F0F1 ATP synthase and one by the combined action of the adenine nucleotide transporter and the phosphate transporter. For each mole of NADH that enters the electron transport chain, 10 moles of protons are pumped out of mitochondria. Thus 10/4 = 2.5 moles of ATP are produced per mole of NADH. Similarly, for each mole of QH2 that enters the electron transport chain six protons are pumped out of mitochondria, yielding 6/4 = 1.5 moles of ATP.

The grand total moles of ATP produced by complete oxidation of glucose to CO2 is thus 4 + 8*2.5 + 4*1.5 = 30 moles!


Inhibition, Regulation and Uncoupling of Oxidative Phosphorylation


Oligomycin, a Streptomyces-produced antibiotic, inhibits ATP synthesis by preventing H+ transport through the F0 domain of the ATP synthase. The plant glycoside atractyloside, and the mold antibiotic bongkrekic acid, inhibit the adenine nucleotide transporter which imports ADP into the mitochondria and exports ATP. Both classes of inhibitor rapidly block oxidative phosphorylation up through the electron transport chain, illustrating that electron transport and ATP synthesis are tightly coupled and operate close to equilibrium. The rate of oxidative phosphorylation is ultimately determined by the availability of ADP, which reflects the energy needs of the cell.










If ATP is not being consumed by the cell, available ADP is depleted by conversion to ATP, causing the F0F1 ATP synthase to stop rotating. Electron transport continues, and the proton gradient grows, until the cost of pumping one more proton out of the mitochondria equals the energy released by the transfer of electrons from NADH to O2. At this point, net electron transport stops; the free energy for the overall process of electron flow coupled to proton pumping has become zero. Oxygen is no longer consumed; NADH accumulates in the mitochonrial matrix and NAD+ is depleted. The lack of NAD+ eventually brings the TCA cycle to a halt.

Tight coupling of electron transport and phosphorylation in mitochonria is disrupted by dinitrophenol (DNP) and other acidic aromatic compounds. DNP has a pKA of ~4 in water. Both its protonated and deprotonated forms are partially soluble in the hydrocarbon core of the inner mitochondrial membrane. Consequently, it can transport protons from the cytosol to the mitochondrial matrix by passive diffusion, dissipating the proton gradient with no concomitant production of ATP. Energy is instead released as heat, resulting in profuse sweating and weight loss in animals administered the compound. This short-circuiting of the mitochondrial proton gradient can be biologically useful. Hibernating animals, some newborn animals including humans, and mammals adapted to cold have a special type of fat called brown adipose tissue. A high density of mitochondria give this tissue its characteristic color. Brown adipose tissue contains thermogenin (also called uncoupling protein), a proton channel in the inner mitochochondrial membrane that opens in response to hormonal signals. The free flow of protons into the mitochondrial matrix produces heat, maintaining body temperature.

Experimental use of DNP as a diet pill in the late 1920’s is described by E. Racker in his historical account A new Look at Mechanisms in Bioenergetics, Academic Press (1976), pg. 155:











Clinical Correlations




II. The Pentose Phosphate Pathway

The pentose phosphate pathway is a second metabolic pathway for complete oxidation of glucose to CO2. It serves two primary purposes: (a) to produce the five carbon sugar ribose which forms the backbone of DNA (b) to generate NADPH reducing equivalents for reductive biosynthesis (for example, for the synthesis of triacylglycerol fat stores in adipose tissue). NADH and NADPH differ only by the presence of a phosphate group on the 3’ alcohol of the adenine moiety, and have similar reduction potentials. However, the two molecules have very different metabolic fates. Almost all NADH is oxidized by the respiratory chain to generate ATP, while NADPH serves primarily as a hydride source for biochemical transformations in the cytosol. The ratios of oxidized to reduced NADH and NADPH in cells are generally different. In the cytosol of a liver cell from a well-fed rat, [NADP+]/[NADPH] = 0.014 while [NAD+]/[NADH]=700. The presence of the additional phosphate group allows enzymes to distinguish between NADH and NADPH as substrates.























The pentose phosphate pathway consists of two independent parts: the oxidative branch and the nonoxidative branch. The oxidative branch produces NADPH equivalents from NADP+ through the oxidation of glucose. The nonoxidative branch interconverts five-carbons sugars with the three and six carbon sugars used by glycolysis. Depending on the metabolic needs of the cell, the two branches may be active together or independently.


Oxidative Branch

Glucose 6-phosphate + 2 NADP+ + H20 --->

Ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2

Glucose 6-phosphate is first oxidized to 6-phosphogluconolactone by glucose 6-phosphate dehydrogenase, generating one NADPH equivalent (cf. glyceraldehyde 3-phosphate dehydrogenase). The lactone is hydrolyzed by lactonase to 6-phosphogluconate. The C3 alcohol of 6-phosphogluconate is subsequently oxidized to the ketone by 6-phosphogluconate deghydrogenase generating a second equivalent of NADPH. Finally, the b-keto acid is decarboxylated to ribulose 5-phosphate by a retro-aldol mechanism (cf. isocitrate dehydrogenase).














The first step of the oxidative branch is rate limiting and essentially irreversible. It is regulated by the NADP+/NADPH ratio in the cytosol of the cell.


Nonoxidatative Branch

The nonoxidative branch of the pentose phophate pathway interconverts three five carbon sugars ribulose 5-phosphate, xylulose 5-phosphate and ribose 5-phosphate, and rearranges five carbon sugars into fructose 6-phosphate and glyceraldehyde 3-phosphate. These carbon gymnastics require no reduction reactions, no oxidation reactions, and no input of energy. They are equilibrium processes. The C5 sugars are interconverted by the enzymes phosphopentose isomerase and phosphopentose epimerase. Thus ribulose 5-phospate, the product of the oxidative branch, is rapidly equilibrated with cellular pools of xylulose 5-phosphate and ribose 5-phosphate.
















The enzymes transketolase and transaldolase catalyze the rearrangement of three C5 sugars to two equivalents of fructose and one equivalent of glyceraldehyde. The stoichiometry of the overall reaction is:

2 Xylulose 5-phosphate + Ribose 5-phospate --->

2 Fructose 6-phosphate + Glyceraldehyde 3-phospate

The reaction proceeds in three steps through the intermediate sugars sedoheptulose 7-phosphate and erythrose 4-phosphate.

The two transformations catalyzed by transketolase move acyl carbanion fragments, which we previously encountered in the pyruvate dehydrogenase reaction. Nature uses the same chemistry here; the acyl carbanion fragments are stabilized by the catalytic cofactor thiamine pyrophosphate (TPP), which acts as an electron sink.




















The transformation catalyzed by transaldolase moves an a-keto carbanion fragment, which we previously encountered in the aldolase reaction. Nature uses the same chemistry here; the a-keto carbanion is stabilized by a protonated Schiff base formed with a lysine on the enzyme.


Configurations of the Pentose Phosphate Pathway

The pentose phosphate pathway can be configured in several different ways to meet different metabolic demands of the cell. The reaction stoichiometries for two limiting cases are provided; it would be a useful exercise for you to derive them given what you know about glycolysis and the pentose phosphate pathway.

1. Ribose 5-phosphate is required, but not NADPH. This situation arises when a cell duplicates the DNA of its genome in preparation for cell division. One molecule of glucose 6-phosphate is run forward through glycolysis to produce two equivalents of glyceraldehyde 3-phosphate. The glyceraldehyde 3-phosphate and four additional moles of glucose 6-phosphate are subsequently run backwards through the non-oxidative branch of the pentose phospate pathway.

5 Glucose 6-phosphate + ATP ---> 6 Ribose 5-phosphate + ADP + H+

2. NADPH is required but not ribose. This situation arises in adipose tissue when it produces triacyglycerol fat stores. Six moles of glucose 6-phosphate are run forward through the oxidative and nonoxidative branches of the pentose phosphate pathway. The products are run backwards through glycolysis to regenerate five moles of glucose 6-phosphate. Overall, one mole of glucose 6-phosphate is oxidized completely to CO2.

Glucose 6-phosphate + 12 NADP+ + 7 H2O --->

6 CO2 + 12 NADPH + 12 H+ + Pi


Clinical Correlations

III. Gluconeogenesis

During periods of extended fasting, the body’s supplies of glucose are depleted. Brain, red blood cells, kidney medulla, lens, cornea, testis and a number of other tissues rely on glucose as their primary energy source. To help these tissues survive during conditions of glucose depletion, the liver and the cortex of the kidney synthesize glucose from noncarbohydrate precursors in a process called gluconeogenesis. The Cori cycle provides an example of this relationship between tissues.













Conversion of glucose to lactate by glycolysis constitutes the primary source of ATP in red blood cells, which lack mitochondria. Lactate exported into the bloodstream by red blood cells is taken up by the liver, converted back into glucose, and returned to the bloodstream to feed glucose-requiring tissues. Thus part of the metabolic load of red blood cells is carried by the liver. A similar cycle obtains between the liver and muscle tissue under anaerobic conditions, such as during vigorous exercise.


Bypass Reactions of Gluconeogenesis


















In order to synthesize glucose from smaller precursors, the liver must reverse the transformations carried out by glycolysis. However, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are associated with large negative free energies and are essentially irreversible. To accomplish gluconeogenesis, the activities of these enzymes must be blocked, and reactions that bypass the three steps (rather than directly reverse them) must be catalyzed. Glucose 6-phosphatase and fructose 1,6-bisphosphatase hydrolyze the high energy phosphoryl bonds present in glycerol 6-phosphate and fructose 1,6-bisphosphate to produce glucose. Note that these phosphatases do not reverse the reactions catalyzed by hexokinase and phosphofructokinase because ATP is not regenerated.

Pyruvate kinase is bypassed in two steps by the enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase. As we have already seen, pyruvate carboxylase resides in the mitochondrial matrix, requires the catalytic cofactor biotin, and converts pyruvate to oxaloacetate at the expense of one high-energy phosphoryl bond of ATP.

Oxaloacetate produced by pyruvate carboxylase is reduced to malate, exported from the mitochondrial matrix by the dicarboxylate transporter, and is reoxidized to oxaloacetate in the cytosol. Phosphenolpyruvate carboxykinase simultaneously decarboxylates and phosphorylates oxaloacetate to generate phosphoenolpyruvate. GTP is used as the phosporyl donor. Decarboxylation drives this reaction, which would otherwise be endergonic (cf. the pyruvate kinase reaction).











The stoichiometry for gluconeogenesis from pyruvate is:

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O --->

Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+

By contrast, the stoichiometry for conversion of glucose to pyruvate by glycolysis is:

Glucose + 2 ADP + 2 Pi + 2 NAD+ --->

2 Pyruvate + 2 ATP + 2 GTP + 2 NADH + 2 H2O

In cycling from glucose ---> pyruvate ---> glucose, four high-energy phosphate bonds are hydrolyzed. This expenditure of energy is required to turn an energetically unfavorable process (the reversal of glycolysis, DG°’ = +20 kcal/mol) into a favorable one (gluconeogenesis, DG°’ = -9 kcal/mol).



In liver, hexokinase is replaced by an enzyme isoform called glucokinase. Glucokinase has a higher Km for glucose than does hexokinase, and also has a higher Vmax. These properties allow glucokinase to phosphorylate large quantities of glucose during periods of high blood glucose concentration (for example, shortly after a carbohydrate-rich meal). Glucokinase also differs from hexokinase because it is regulated by the glucokinase inhibitor protein in response to fructose 6-phospate/fructose 1,6-bisphosphate levels. When bound to fructose 6-phosphate, the glucokinase inhibitor protein sequesters and inactivates glucokinase; when bound to fructose 1,6-bisphosphate it does not associate with glucokinase.


Substrates for Gluconeogenesis

The master regulator of gluconeogenesis is glucagon. A primary effect of glucagon is to activate lipases in adipose tissue, promoting release of fatty acids into the bloodstream. These fatty acids are broken down in the mitochondria of liver, resulting in high concentrations of acetyl CoA. The acetyl CoA feeds the TCA cycle and generates ATP by oxidative phosphorylation. Recall, however, that animals are unable to recycle acetyl Co A into larger carbon units. Thus acetyl CoA, the major breakdown product of fat, can not be used as a carbon source for gluconeogenesis.

The primary carbon substrates for gluconeogenesis in the liver are lactate, amino acids, glycerol and succinyl CoA. Lactate is produced by tissues that utilize the Cori cycle, and is oxidized to pyruvate for entry into gluconeogenesis. Amino acids are produced by the break down of muscle, and are converted to a-keto acids for gluconeogenesis by a pyridoxal-phosphate dependent transamination reaction (transamination will be revisited in the lectures on amino-acid metabolism). Glycerol is generated from triacylglycerols (fat storage molecules) by triacylglycerol lipases. It is phosphorylated by triose kinase and oxidized to dihydroxyacetone phosphate by glycerol 3-phophate dehydrogenase for entry into gluconeogenesis. Finally, succinyl CoA is a break-down product of odd-chain fatty acids and is oxidized to oxaloacetate for gluconeogenesis by the enzymes of the TCA cycle.


Glycolysis and Gluconeogenesis are Reciprocally Regulated

To accomplish gluconeogenesis, the enzymes catalyzing irreversible steps of glycolysis must be shut down, and the enzymes catalyzing bypass reactions must be activated. This metabolic reprogramming is orchestrated by the peptide hormone glucagon which directs protein phosphorylation and indirectly alters concentrations of the allosteric regulators acetyl CoA and fructose 2,6-bisphosphate. Glucagon acts directly by activating adenylate cyclase, which elevates levels of the second messenger cAMP. cAMP activates protein kinase A, which phosphorylates and inactivates pyruvate kinase. In addition, protein kinase A phosphorylates the bifunctional enzyme 6-phosphofructo-2 kinase/fructose 2,6 bisphosphatase. Phosphorylation of this enzyme inhibits the kinase and activates the phosphatase, resulting in drastically lowered levels of fructose 2,6-bisphosphate. As discussed above, glucagon indirectly alters the levels of acetyl CoA in liver by effecting release of fatty acids by adipose tissue. Three events, phosphorylation of pyruvate kinase, reduction of [fructose 2,6-bisphosphate], and elevation of [acetyl CoA] are primarily responsible for the reciprocal control of gluconeogenesis and glycolysis as follows:


Shutdown of Glycolysis:

Pyruvate Kinase Phosporylated and inactivated

by protein kinase A

Pyruvate Dehydrogenase Phosphorylated and inactivated

b/c of high [acetyl CoA]

Phosphofructokinase Allosterically downregulated b/c of low

[fructose 2,6-bisphosphate]

Glucokinase Inhibited by glucokinase inhibitor protein b/c of high [fructose 6-phosphate] and

low [fructose 1,6-bisphosphate]


Activation of Bypass Enzymes:

PEP Carboxykinase Transcriptionally activated

Pyruvate Carboxylase Allosterically activated by high [acetyl CoA]

Fructose 1,6-bisphosphatase Allosterically activated b/c of low

[fructose 2,6-bisphosphate]

Glucose 6-phosphatase Transcriptionally activated

Increased concentrations of acetyl CoA reciprocally regulate the activities of pyruvate dehydrogenase and pyruvate carboxylase. Similarly, decreased concentrations of fructose 2,6-bisphosphate reciprocally regulate the activities of phosphofructokinase and fructose 1,6-bisphosphatase, resulting in accumulation of fructose 6-phosphate and depletion of fructose 1,6-bisphosphate. This alteration of the fructose 6-phosphate/fructose 1,6-bisphosphate ratio causes glucokinase inhibitor protein to sequester and inhibit glucokinase.

Phosporylation and allosteric control operate on the minute to hour timescale. On the hour to day timescale, elevated levels of glucagon stimulate the transcription of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase.





Clinical Correlations