Bioenergetics and Oxidative Metabolism

ATP is the central metabolic currency

Oxidation of metabolic fuels is essential to life. In higher organisms, fuels such as carbohydrates and lipids are metabolized to carbon dioxide and water, generating a central metabolic currency, adenosine triphosphate (ATP). Most metabolic energy is produced by oxidation-reduction (redox) reactions in mitochondria. The regulation of energy metabolism is no small feat, because warm-blooded animals have such variable demands for energy from such processes as thermogenesis at low temperatures, stimulation of ATP synthesis during stress, and coupling of ATP synthesis with the rate of respiration during work and exercise. This chapter will provide an introduction to the concept of free energy, oxidative phosphorylation and the transduction of energy from fuels into useful work. The pathways and specific molecules through which electrons are transported to oxygen and the mechanism of generation of ATP will be described and related to the structure of the mitochondrion, the powerhouse of the cell and the major source of cellular ATP. Lastly, these biochemical processes will be applied to human health and disease.

The basal metabolic rate is a measure of the total daily energy expenditure by the body at rest

Virtually all of the reactions in the body are exothermic, and the sum of all reactions at rest is called the basal metabolic rate (BMR), which can be measured by two basic methods: direct calorimetry, where the total heat liberated by an animal is measured over time, and indirect calorimetry, where the BMR is calculated from the quantity of oxygen consumed, which is directly related to the BMR. Adult men (70 kg) have a BMR of about 7500 kJ (1800 kcal) and women about 5400 kJ (1300 kcal) per day; the BMR may vary by a factor of two between individuals, depending on age, sex, body mass and composition. Heat production by mitochondria accounts for the largest portion of the BMR. The BMR is measured under controlled conditions: after an 8-hour sleep, in the reclining position, in the postabsorptive state, typically after a 12-hour fast.

Another measure often used is the RMR, or resting metabolic rate, which is virtually the same as the BMR but measured under less restrictive conditions. The RMR is a measure of minimum energy expenditure at rest; it is typically about 70% of total daily energy expenditure. Exercise scientists frequently use the term MET, metabolic equivalent task, as a measure of energy expenditure at rest. Slow to vigorous walking is a 2–4 MET/h activity; vigorous running on a treadmill may consume more than 15 MET/h.

The direction of a reaction depends on the difference between the free energy of reactants and products

The Gibbs' free energy (ΔG) of a reaction is the maximum amount of energy that can be obtained from a reaction at constant temperature and pressure. The units of free energy are kcal/mol (kJ/mol). It is not possible to measure the absolute free energy content of a substance directly, but when reactant A reacts to form product B, the free energy change in this reaction, ΔG, can be determined.

For the reaction A → B:

where G_A and G_B are the free energy of A (reactant) and B (product), respectively. All reactions in biological systems are considered to be reversible reactions, so that the free energy of the reverse reaction, B → A, is numerically equivalent, but opposite in sign to that of the forward reaction.

If there is a greater concentration of B than of A at equilibrium, i.e. Keq > 1, the conversion A → B is favorable – that is, the reaction tends to move forward from a standard state in which A and B are present at equal concentrations. In this case, the reaction is said to be a spontaneous or exergonic reaction, and the free energy of this reaction is defined as negative: that is, ΔG < 0, indicating that energy is liberated by the reaction. Conversely, if the concentration of A is greater than that of B at equilibrium, the forward reaction is termed unfavorable, nonspontaneous or endergonic, and the reaction has a positive free energy: that is, when starting concentrations are equal, B tends to form A, rather than A to form B. In this case, energy input would be required to push the reaction A → B forward from its equilibrium position to the standard state in which A and B are present at equal concentrations. The total free energy available from a reaction depends on both its tendency to proceed forward from the standard state (ΔG) and the amount (moles) of reactant converted to product.

The free energy of metabolic reactions is related to their equilibrium constants by the Gibbs' equation

Thermodynamic measurements are based on standard-state conditions where reactant and product are present at 1 molar concentrations, the pressure of all gases is 1 atmosphere and the temperature is 25°C (298K). Most commonly, the concentrations of reactants and products are then measured after equilibrium is attained. Standard free energies are represented by the symbol ΔG° and biological standard free energy change by ΔG°′, with the accent symbol designating pH 7.0. The free energy available from a reaction may be calculated from its equilibrium constant by the Gibbs' equation:

where T is absolute temperature (Kelvin), lnK′_eq is the natural logarithm of the equilibrium constant for the reaction at pH 7, and R is the ideal gas constant:

Several common metabolic intermediates that you will encounter in your studies are listed in Table 9.2, along with the equilibrium constants and free energies for their hydrolysis reactions. Those intermediates with free energy changes equal to or greater than that of ATP, the central energy transducer of the cell, are considered to be high-energy compounds, and generally have either anhydride or thioester bonds. The lower-energy compounds listed are all phosphate esters and, in comparison, do not yield as much free energy on hydrolysis. The hydrolysis reaction of glucose-6-phosphate (Glc-6-P) is written as:

This reaction has a negative free energy and occurs spontaneously. The reverse reaction, synthesis of Glc-6-P from glucose and phosphate, would require input of energy.

ATP is a product of catabolic reactions and a driver of biosynthetic reactions

Living systems must transfer energy from one molecule to another without losing all of it as heat. Some of the energy must be conserved in a chemical form in order to drive nonspontaneous biosynthetic reactions. In fact, nearly half of the energy obtained from the oxidation of metabolic fuels is channeled into the synthesis of ATP, a universal energy transducer in living systems. ATP is often referred to as the common currency of metabolic energy, because it is used to drive so many energy-requiring reactions. ATP consists of the purine base adenine, the five-carbon sugar ribose, and α, β, and γ phosphate groups (Fig. 9.2). The two phosphoanhydride linkages are said to be high-energy bonds, because their hydrolysis yields a large negative change in free energy. When ATP is used for metabolic work, these high-energy linkages are broken and ATP is converted to ADP or to AMP.

The free energy of a high-energy bond, such as the phosphate anhydride bonds in ATP, can be used to drive or push forward reactions that would otherwise be unfavorable. In fact, nearly all biosynthetic pathways are thermodynamically unfavorable, but are made favorable by coupling various reactions with hydrolysis of high-energy compounds. For example, the first step in the metabolism of glucose is the synthesis of Glc-6-P (Fig. 3.4). As shown in Table 9.2, this is not a favorable reaction: the hydrolysis of Glc-6-P (ΔG°′ = −13.8 kJ/mol or −3.3 kcal/mol) is the favored reaction. However, as shown below, the synthesis of Glc-6-P (reaction I) can be energetically coupled to the hydrolysis of ATP (reaction II), yielding a ‘net reaction’ III that is favorable for synthesis of Glc-6-P:

This is possible because of the high free energy or ‘group transfer potential’ of ATP. The physical transfer of the phosphate from ATP to glucose occurs in the active site of a kinase enzyme, such as glucokinase. This motif, in which ATP is used to drive biosynthetic reactions, transport processes or muscle activity, occurs commonly in metabolic pathways.

NAD⁺, FAD, and FMN are the major redox coenzymes

The major redox coenzymes involved in transduction of energy from fuels to ATP are nicotinamide adenine dinucleotide (NAD⁺), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) (Fig. 9.4). During energy metabolism, electrons are transferred from carbohydrates and fats to these coenzymes, reducing them to NADH, FADH₂ and FMNH₂. In each case, two electrons are transferred, but the number of protons transferred differs. NAD⁺ accepts a hydride ion (H^–) that consists of one proton and two electrons; the remaining proton is released into solution. FAD and FMN accept two electrons and two protons.

The oxidation of reduced nucleotides by the electron transport system produces a large amount of free energy. When the oxidation of 1 mole of NADH is coupled to the reduction of 0.5 mole of oxygen to form water, the energy produced is theoretically sufficient to synthesize 7 moles of ATP:

Dividing 220 kJ/mol of ΔG°′ available from oxidation of NADH by ΔG°′ 30.5 required for synthesis of ATP yields, theoretically, 7 mol ATP/mol NADH. As discussed below, the actual yield is closer to 2.5 mol ATP/mol NADH oxidized.

The free energy of oxidation of NADH and FADH₂ is used by the electron transport system to pump protons into the intermembrane space. The energy produced when these protons reenter the mitochondrial matrix is used to synthesize ATP. This process is known as oxidative phosphorylation (see Fig. 9.3)

The mitochondrial electron transport chain transfers electrons in a defined multi-step sequence from reduced nucleotides to oxygen

The entire electron transport system, also known as the electron transport chain or respiratory chain, is located in the inner mitochondrial membrane (Fig. 9.5). It consists of several large protein complexes and two small, independent components – ubiquinone and cytochrome c. The protein components are very complex; complex I, for example, which accepts electrons from NADH, contains at least 46 subunits. Each step in the electron transport chain involves a redox reaction where electrons are transferred from components with more negative reduction potentials to components with more positive reduction potentials. Electrons are conducted through this system in a defined sequence from reduced nucleotide coenzymes to oxygen, and the free energy changes drive the transport of protons from the matrix into the intermembrane space via the three proton pumps. After each step, the electrons are at a lower energy state.

Electrons are funneled into the electron transport chain by several flavoproteins

There are four flavoproteins in the electron transport chain: Complex I contains FMN and the other three contain FAD. These flavoproteins all reduce the small, lipophilic molecule ubiquinone (Q or coenzyme Q₁₀), at the beginning of the common electron transport pathway, consisting of Q, complex III, cytochrome c, and complex IV.

Protons are pumped from the matrix into the intermembrane space by complexes I, III, and IV. Oxygen (O₂) is the final electron acceptor at the end of the chain, and it is reduced to two water molecules by the transfer of four electrons from complex IV and four protons from the mitochondrial matrix compartment.

The efficiency of oxidative phosphorylation can be measured by dividing the amount of phosphate incorporated into ADP by the amount of atomic oxygen reduced. One atom of oxygen is reduced by two electrons (one electron pair).

For each pair of electrons transported through complexes I, III or IV, a sufficient number of protons is pumped by each complex for the synthesis of approximately one mole of ATP/complex. If electron transport begins with an electron pair from NADH, approximately 2.5 moles of ATP are synthesized, whereas an electron pair from any of the other three FADH₂-containing flavoproteins yields about 1.5 moles of ATP, because the proton-pumping capability of complex I is bypassed.

Electron shuttles are required for mitochondrial oxidation of NADH produced in the cytoplasmic compartment

NADH is produced in the cytosol during carbohydrate metabolism. NADH cannot cross the inner mitochondrial membrane, and therefore it cannot be oxidized by the electron transport system. Two redox shuttles permit the oxidation of cytosolic NADH without its physical transfer into the mitochondrion. A characteristic feature of these shuttles is that they are powered by cytoplasmic and mitochondrial isoforms of the same enzyme, which catalyze opposing reactions on opposite sides of the membrane. The glycerol-3-P shuttle is the simpler of the two (Fig. 9.7, top). It transfers the electrons of NADH from the cytoplasm to the mitochondrion by reducing FAD to FADH₂. Cytoplasmic glycerol-3-P dehydrogenase catalyzes reduction of dihydroxyacetone-P (DHAP) with NADH to glycerol-3-P, regenerating NAD⁺. The cytoplasmic glycerol-3-phosphate is oxidized back to DHAP by another glycerol-3-phosphate dehydrogenase isoform facing the outer surface of the inner mitochondrial membrane; this enzyme is a flavoprotein in which FAD is reduced to FADH₂. The electrons are then transferred to the common pathway via ubiquinone. Because the electrons are transferred to FAD, the yield of ATP from cytoplasmic NADH by this pathway is approximately 1.5 moles, rather than the 2.5 moles available from mitochondrial NADH via the NADH-Q reductase complex (complex I).

Many cells, e.g. in skeletal muscle, use the glycerol-3-P shuttle, but heart and liver rely on the malate–aspartate shuttle (Fig. 9.7, bottom), which yields 2.5 moles of ATP per mole of NADH. This shuttle is more complicated, because the substrate, malate, can cross the inner mitochondrial membrane, but the membrane is impermeable to the product, oxaloacetate – there is no oxaloacetate transporter. The exchange is therefore accomplished by interconversion between α-keto- and α-amino acids, involving cytoplasmic and mitochondrial glutamate and α-ketoglutarate, and isozymes of glutamate-oxaloacetate transaminase (aspartate aminotransferase).

Ubiquinone tranfers electrons from flavoproteins to complex III

Ubiquinone is so named because it is ubiquitous in virtually all living systems. It is a small, lipid-soluble compound found in the inner membrane of animal and plant mitochondria and in the plasma membrane of bacteria. The primary form of mammalian ubiquinone contains a side chain of 10 isoprene units and is often called CoQ₁₀. It diffuses within the inner membrane, accepts electrons from the four major mitochondrial flavoproteins, and transfers them to complex III (QH₂–cytochrome c reductase). Ubiquinone can carry either one or two electrons (Fig. 9.8) and is also thought to be a major source of superoxide radicals in the cell (see Chapter 37).

Complex III accepts electrons from ubiquinone and pumps four hydrogen ions across the inner mitochondrial membrane

This enzyme complex, also known as ubiquinone–cytochrome c reductase or QH₂–cytochrome c reductase, oxidizes ubiquinone and reduces cytochrome c. Reduced ubiquinone funnels electrons that it gathers from mitochondrial flavoproteins and transfers them to complex III. Electrons from ubiquinone are transferred through two species of cytochrome b, to an FeS center, to cytochrome c₁, and finally to cytochrome c. Transport of two electrons to cytochrome c yields sufficient free energy change and protons pumped to synthesize about one mole of ATP. The overall reaction is:

Four protons are pumped during this reaction, two from fully reduced ubiquinone and two from the matrix.

Cytochrome c is a peripheral membrane proteins, shuttling electrons from complex III to complex IV

Cytochrome c, a small heme protein that is loosely bound to the outer surface of the inner membrane, shuttles electrons from complex III to complex IV. Each cytochrome c carries only one electron, so the reduction of O₂ to 2H₂O by complex IV requires four reduced cytochrome c molecules. The binding of cytochrome c to complexes III and IV is largely electrostatic, involving a number of lysine residues on the protein surface. Reduction of ferricytochrome c (Fe³⁺) to ferrocytochrome c (Fe²⁺) by cytochrome c₁ leads to a change in the three-dimensional structure, charge distribution and dipole moment of the protein, promoting transfer of electrons to cytochrome a in complex IV (see Fig. 9.5). In response to oxidative stress and cell injury (Chapter 37), cytochrome c may be released from the inner mitochondrial membrane and leak into the cytosol, inducing apoptosis (cell death).

The ATP synthase complex (complex V) is an example of rotary catalysis

Lining the inner matrix face of the inner membrane of each mitochondrion are thousands of copies of the ATP synthase complex, also called complex V or F₀F₁-ATP synthase (F = coupling factor; see Inhibitors of ATP synthase, below). ATP synthase is also called an ATPase, because it can hydrolyze ATP. ATP synthase consists of two major complexes (Fig. 9.11). The inner membrane component, termed F₀, is the proton-driven motor with the stoichiometry of a, b₂ and c_10–14. The c-subunits form the c-ring, which rotates in a clockwise direction in response to the flow of protons through the complex. Since the γ- and ε-subunits are bound to the c-ring, they rotate with it, inducing large conformational changes in the three-αβ dimers of the F₁ complex. The two β-proteins immobilize the second complex (F₁-ATP synthase).

F₁ has a stoichiometry of α₃, β₃, γ, δ, ε. The major part of F₁ consists of three αβ dimers arranged like slices of an orange, with the catalytic activity residing on the β-subunits. Each 120° rotation of the γ-subunit induces conformational changes in the αβ-dimeric subunits such that the nucleotide-binding sites alternate between three states: the first binds ADP and Pi, the second synthesizes ATP, and the third releases ATP, so each complete turn produces 3ATP. This is known as the binding-change mechanism (Fig. 9.12). Surprisingly, the proton-motive free energy used by ATP synthase is not for ATP synthesis itself, but for its release; when the proton gradient is too low to support ATP release, ATP remains stuck to ATP synthase and further ATP production ceases. ADP and Pi are bound to the complex as soon as ATP leaves. The αβ-dimers are asymmetrical, because each is in a different conformation at any given moment. This complex is a proton-driven motor, and it is an example of rotary catalysis. About three protons are required for the synthesis of each ATP. This complex acts independently of the electron transport chain; addition of a weak acid, such as acetic acid, to a suspension of isolated mitochondria is sufficient to induce the biosynthesis of ATP in vitro.

‘Respiratory control’ is the dependence of oxygen uptake by mitochondria on the availability of ADP

Normally, oxidation and phosphorylation are tightly coupled: substrates are oxidized, electrons are transported, and oxygen is consumed only when synthesis of ATP is required (coupled respiration). Thus, resting mitochondria consume oxygen at a slow rate, which can be greatly stimulated by addition of ADP (Fig. 9.13). ADP is taken up by the mitochondria and stimulates ATP synthase, which lowers the proton gradient. Respiration increases, because the proton pumps are stimulated to reestablish the proton gradient. When the ADP is depleted, ATP synthesis terminates and respiration returns to the original rate. Oxygen uptake declines to the original rate when the concentration of ADP is depleted and ATP synthesis terminates.

Mitochondria can become partially uncoupled if the inner membrane loses its structural integrity. They are said to be ‘leaky’, because protons can diffuse through the inner membrane without involving ATP synthase. This occurs if isolated mitochondria are treated with mild detergents that disrupt the inner membrane, or if they have been stored for a period of time. Such mitochondria are said to be ‘uncoupled’; oxidation proceeds without production of ATP and uncoupled mitochondria lose respiratory control because protons pumped by the electron chain bypass the ATPase and leak unproductively back into the matrix. The P : O ratio declines under these conditions.

The mechanism of respiratory control depends on the requirement for ADP and Pi binding to the ATP synthase complex: in the absence of ADP and Pi, protons cannot enter the mitochondrion through this complex and oxygen consumption markedly decreases, because the proton pumps cannot pump protons against a high proton back-pressure. This happens because the free energy of the electron transport reactions is sufficient to generate a pH gradient of only two units across the membrane. If the pH gradient cannot be discharged for production of ATP, the two pH unit gradient is established and the pumps grind to a halt and stall. The electron transport chain becomes reduced and substrate oxidation and oxygen consumption decrease. A little physical activity, with consumption of ATP and generation of ADP and Pi, opens up the ATPase channels, discharging the proton gradient and activating the electron transport chain, and fuel and oxygen consumption. At a whole-body level, we breathe faster during exercise to provide the additional oxygen needed for increased oxidative phosphorylation.

Uncouplers and uncoupling proteins are thermogenic

Uncouplers of oxidative phosphorylation dissipate the proton gradient by transporting protons back into mitochondria, bypassing the ATP synthase. Uncouplers stimulate respiration, because the system makes a futile attempt to restore the proton gradient by oxidizing more fuel and pumping more protons out of mitochondria. Uncouplers are typically hydrophobic compounds and either weak acids or bases, with pK_a near pH 7. The classic uncoupler, 2,4-dinitrophenol (DNP) (Fig. 9.14), is protonated in solution on the outer, more acidic side of the inner mitochondrial membrane. Because of its hydrophobicity, it may then freely diffuse through the inner mitochondrial membrane. When it reaches the matrix side, it encounters a more basic pH and the proton is released, effectively discharging the pH gradient. Other uncouplers include preservatives and antimicrobial agents, such as pentachlorophenol and p-cresol.

Rotenone inhibits complex I (NADH–Q reductase)

Rotenone, a common insecticide, and some barbiturates (e.g. amytal) inhibit complex I. Because malate and lactate are oxidized by NAD⁺, their oxidation will be decreased by rotenone. However, substrates yielding FADH₂ can still be oxidized, because complex I is bypassed and electrons are donated to ubiquinone from FADH₂. Addition of ADP to a suspension of mitochondria supplemented with malate and phosphate (Fig. 9.15) markedly stimulates oxygen uptake as ATP synthesis occurs. Oxygen uptake is markedly inhibited by rotenone, but when succinate is added, ATP synthesis and oxygen consumption resume until the supply of ADP is exhausted. Rotenone inhibition of complex I causes reduction of all components prior to the point of inhibition, because they cannot be oxidized, whereas those after the point of inhibition become fully oxidized. This is known as a crossover point, and it can be determined spectrophotometrically, because light absorption by respiratory chain components changes according to redox state. Such analyses were used to define the sequence of components in the respiratory chain.

Antimycin A inhibits complex III (QH₂–cytochrome c reductase)

The inhibition of complex III by antimycin A prevents transfer of electrons from either complex I or FADH₂-containing flavoproteins to cytochrome c. In this case, components preceding complex III become fully reduced, and those after it become oxidized. The oxygen uptake curve (Fig. 9.16) shows that the stimulation of respiration by ADP is inhibited by antimycin A, but that the addition of succinate does not relieve the inhibition. Ascorbic acid can reduce cytochrome c, and addition of ascorbic acid restores respiration, illustrating that complex IV is unaffected by antimycin A.

Cyanide and carbon monoxide inhibit complex IV

Azide , cyanide (CN)⁻; and carbon monoxide (CO) inhibit complex IV (cytochrome c oxidase) (Fig. 9.17). Because complex IV is the terminal electron transfer complex, its inhibition cannot be bypassed. All components preceding complex IV become reduced, oxygen cannot be reduced, none of the complexes can pump protons, and ATP is not synthesized. Uncouplers such as DNP have no effect, because there is no proton gradient. Cyanide and carbon monoxide also bind to hemoglobin, blocking oxygen binding and transport (see Chapter 5). In these poisonings, both the ability to transport oxygen and to synthesize ATP are impaired. The administration of oxygen is used for the treatment of such poisonings. It is of interest that sodium azide is the nitrogen source for the inflation of air bags; it may pose an environmental problem if it is accidentally released, i.e. nonexplosively.

ADP is the key feedback regulator of oxidative phosphorylation

The oldest and simplest known mechanism of respiratory control is accomplished by the supply of ADP. This is based on the fact that when added to isolated mitochondria, ADP stimulates respiration and ATP synthesis. When ADP is completely converted to ATP, respiration returns to the initial rate. Oxidative phosphorylation is also tightly coupled to other fundamental pathways such as glycolysis, fatty acid oxidation and the tricarboxylic acid cycle (see Chapters 12, 14 and 15) through feedback regulatory mechanisms. The ratios of NADH/NAD and ATP/ADP feedback on key enzymes, such as PFK-1, pyruvate dehydrogenase and isocitrate dehydrogenase. If, for example, oxidative phosphorylation is switched off because of high ATP, both NADH and ATP will negatively feedback on other extramitochondrial pathways, limiting the flux of fuel to mitochondria. Since oxidative phosphorylation depends on the supply of FADH₂, NADH, ADP, and Pi as well as the ATP/ADP ratio, magnitude of the membrane potential, uncoupling and hormonal factors, its modes of regulation are clearly complex.