The Electron-Transfer Chain and Oxidative Phosphorylation

The Electron-Transfer Chain and Oxidative Phosphorylation The Mitochondrion - Scene of the Action 1.1 The inner mitochondrial membrane compartmentalizes metabolic functions. Although mitochondria in different kinds of eukaryotic cells vary widely in size, shape, and number, most are about 1-2 µm long and 0.5-1.0 µm in diameter. The outer membrane is smooth and continuous without obvious foldings. The inner membrane is also continuous, but it is folded into shelf-like cristae that greatly increase the surface of the inner membrane. The lumen between the inner and outer membranes is called the intermembrane space, and the compartment contained within the inner membrane is the matrix. Most mitochondrial proteins are nuclear gene products imported from the cytosol. Mitochondrial Outer Membrane. The outer membrane is less dense than the inner membrane. This density difference is due to the fact that the outer membrane contains twice as much phospholipid as the inner membrane. The inner and outer membranes also differ in their composition of lipids and proteins. Cardiolipin, a major phospholipid component of the inner membrane, is absent from the outer membrane. Phosphatidylinositol and cholesterol are important constituents of the outer membrane, but are absent from the inner membrane. The outer membrane contains a number of enzymes concerned with phospholipid biosynthesis. However, the major protein in the outer membrane is porin. This transmembrane protein forms pores or channels about 20 to 30 Å in diameter, creating a "molecular sieve" that permits free passage of most low-mw solutes. No proteins in the outer membrane are coded by any genes in the mitochondrial genome. Mitochondrial Intermembrane Space. Several important enzymes coded by nuclear genes are found in the intermembrane space. Mitochondrial Inner Membrane. The inner membrane is impermeable to all ions and hydrophilic solutes for which specific transport systems are lacking. Molecular oxygen, 58

however, is lipophilic and readily diffuses across the inner membrane. This membrane contains some 80 proteins involved in electron transport and oxidative phosphorylation. In addition, another 30 or so transporters are responsible for the exchange of ADP, ATP, Pi, and fuel molecules between the matrix and the cytosolic compartment. Together, these proteins comprise about 75% of the membrane. Mitochondrial genome encodes about a fifth of the membrane proteins involved in electron transport and ATP synthesis. Mitochondrial Matrix. The matrix is home to all the soluble enzymes of the citric acid cycle and the pathways for the oxidation of fatty acids and amino acids. These pathways generate NADH and FADH 2 for oxidative phosphorylation. In addition, the matrix houses the genetic machinery of the mitochondrion. 1.2 The mitochondrion possesses its own unique genetic system. The genes present in the mitochondrial genome code for specific components required for mitochondrial function and these tend to be highly conserved phylogenetically. The expression of these genes relies on RNA and protein synthesizing systems unique to this organelle. Mammalian mitochondrial genomes, including the human, are all circular DNA duplexes of 16.5 kilobase pairs. Approximately one-fifth of the genes needed for oxidative phosphorylation are in the mitochondrial genome. However, the vast majority of membrane proteins and all the soluble enzymes of the mitochondrion are nuclear gene products. The proteins encoded by the mitochondrial genome are specific components of the respiratory electron transfer chain and the ATP synthase. Although the events leading to the evolution of modern chloroplasts and mitochondria occurred a billion or so years ago, these proteins are highly conserved and exhibit a high degree of sequence identity to their counterparts in bacteria and chloroplasts. In 1970 Lynn Margulis published an important book entitled Origin of Eukaryotic Cells that championed the hypothesis that eukaryotic organelles had been acquired by early eukaryotic cells in a series of endosymbiotic events. A spirited debate ensued as to whether chloroplasts and mitochondria were indeed of symbiotic origin or whether they arose within eukaryotic cells in the process of cellular evolution. The debate took a distinct turn in favor of the endosymbiotic hypothesis in the 1980s when comparative data on gene sequences for rrnas became available. These molecules possess highly conserved as well as variable regions and are components of all protein synthesizing systems. Analysis of rrna sequence data has now provided convincing evidence that evolutionary histories of the nuclear and organelle genomes are certainly different. Moreover, the chloroplast and mitochondrial genomes most certainly arose from separate bacterial lineages. Therefore the analysis of rrna gene sequences appears to have furnished indisputable evidence in support of the endosymbiotic hypothesis. The vast majority of mitochondrial proteins are encoded in nuclear DNA, including those proteins involved in mitochondrial DNA replication, transcription, and translation. All the proteins in the matrix, intermembrane space, and the outer membrane, along with those inner membrane proteins encoded by nuclear DNA, are synthesized in the cytosolic compartment and transported to their specific mitochondrial locations by means of signal peptide sequences and molecular chaperones. (Chaperons are proteins that mediate the correct assembly of the polypeptide chains). 59

Oxidative Phosphorylation: How Electrons Meet Oxygen to Drive ATP Generation 2.1 ATP synthesis by oxidative phosphorylation supplies most of the energy for cell function in animals. Molecular oxygen is essential for almost all the cells of the body. Without an adequate and continuing supply of O 2, the cells deteriorate and die. The speed at which they die depends a great deal on their makeup and function. For example, the cells of the brain and nervous system may die in four to six minutes after being deprived of O 2, and since the brain is the master organ of the body, the death of its cells causes other body processes to stop. This vital process cellular respiration takes place in the mitochondrion. Here the oxidative reactions of the citric acid cycle and fatty acid oxidation produce the electrons, that when they travel through the respiratory chain to oxygen, provide the energy required to regenerate ATP. Thus, the oxidation of carbohydrates and fats can be viewed simply as the removal of electrons (plus H + ions) from these fuel molecules. The electrons are then passed through the coenzymes and iron atoms of the enzymes of the electron transfer chain. Eventually the electrons are transferred to molecular oxygen, yielding H 2 O. O 2 + 4 e + 4 H + 2 H 2 O The formation of water, the last reaction in respiration, is catalyzed by cytochrome oxidase, one of the fifteen or more enzymes that comprise the electron transfer chain in the inner mitochondrial membrane. The cytochrome oxidase reaction accounts for more than 95% of the molecular oxygen used by the body. The remainder is used for numerous biosynthetic reactions, for example, the hydroxylation of lysine residues during collagen biosynthesis. Oxidative phosphorylation consists of two tightly coupled processes: (1) the generation of a proton electrochemical gradient across the mitochondrial inner membrane by the respiratory chain and (2) the use of the proton gradient by the mitochondrial ATP synthase to catalyze the phosphorylation of ADP (Fig. 2). Although the oxidation of carbohydrates and fats can be viewed as combustion, the mechanism is clearly different. In fact, none of the oxygen atoms in the CO 2 produced during the oxidation of fuels in the mitochondria come from molecular oxygen. The O atoms in CO 2, are furnished by either the substrate itself or water molecules. The strategy of respiration is simply that electrons are stripped from energy-rich fuel molecules by the reactions of the citric acid cycle and fatty acid oxidation and passed to NAD + and FAD. The reduced coenzymes in turn deliver the electrons to the electron transfer chain. Carbon dioxide is formed by the decarboxylation of carboxylic acids generated by the reactions of the citric acid cycle. 60

Figure 2. The proton circuit of mitochondria. The generation of ATP during oxidative phosphorylation is linked to a proton current across the mitochondrial inner membrane. Protons are pumped out of the matrix as electrons pass through the electron transfer chain. This establishes a proton concentration gradient and an electric potential across the inner membrane. ATP is formed by the enzymic action of ATP synthase as protons re-enter the matrix. 2.2 Oxidative phosphorylation begins with the removal of electrons from NADH and FADH 2 by the respiratory electron transfer chain, which eventually dumps them on molecular oxygen. The electron transport chain is housed within the inner membrane of the mitochondrion and is organized as a linked series of electron transfers from reduced coenzymes to molecular oxygen. This process is organized as a complex set of cog-wheels or stoichiometrically linked redox reactions. Neither the exact stoichiometry nor the precise sequence of intermediates is fully understood at this time. The reactions of respiration can be looked on either as the transfer of electrons, or the transfer of reducing equivalents. Thus, we can view the overall reactions of oxidative phosphorylation as the transfer of hydrogen from NADH and FADH 2 to oxygen: NADH + H + + 1/2 O 2 NAD + + H 2 O and FADH 2 + O 2 FAD + H 2 O Alternatively, we can consider these reactions in terms of electron fluxes NADH + H + NAD + + 2 e + 2 H + and FADH 2 FAD + 2 e + 2 H + 61

Oxidative phosphorylation, therefore, occurs when the electrons carried by the reduced coenzymes NADH and FADH 2 are transferred to molecular oxygen via the electron transfer chain. 1/2 O 2 + 2 e + 2 H + H 2 O If we consider a hydrogen atom as an electron and H + (e + H + = H. ), we can view the overall reaction for the oxidation of NADH or FADH 2 as the transfer of hydrogen atoms, that is, reducing equivalents, to molecular oxygen via the electron transfer chain. The difference, however, is that the explosive reaction that occurs when molecular hydrogen is ignited in air does not occur when the reaction is catalyzed by the electron transfer chain. Instead, the reaction takes place in a non-explosive manner. Furthermore, this reaction cannot take place unless ADP and Pi are being imported from the cytosolic compartmentthat is, respiration is coupled to A TP generation. Thus, oxidative phosphorylation provides a mechanism to "trap" free energy as adenylate energy. The removal of electrons from fuel molecules requires that the electron acceptors, NAD + and FAD, be regenerated as fast as they are reduced. In other words, the mitochondrial oxidation of fuel molecules is coupled to two redox cycles, specifically, the NAD and FAD cycles in the matrix compartment. As NADH and FADH 2 are reoxidized by the electron transfer chain, electrons flow through a series of tightly linked redox reactions. Starting with NADH, removal of electrons reduces the first electron carrier in the chain. That carrier is reoxidized when it passes the electrons on to the next component of the chain, and so on. Thus each electron carrier cycles continuously between its reduced and oxidized states. Some oxidized carriers accept two electrons, others only one at a time. Eventually, four electrons are gathered by cytochrome c oxidase, which then catalyzes the four-electron reduction of O 2 to two H 2 O. The four-electron reduction of O 2 is necessary to avoid the formation of oxygen free radicals. These highly reactive oxygen species, were they to "escape" within the cell, could cause considerable damage to proteins, membranes, and nucleic acids. Any disruption in electron transfer brings the entire catalytic sequence to a complete halt. This accounts for the highly toxic actions of agents, which act on specific components of the electron transfer chain and block their recycling. These include such famous poisons as rotenone, which inhibits complex I (see below) and antimycin, an inhibitor of complex III. Cytochrome c oxidase is inhibited by azide, cyanide, and carbon monoxide. 2.3 A " chain" of fifteen or more electron carriers spans 1,070 mv difference between NADH and oxygen. As depicted in Figure 3, the respiratory chain includes four multimeric complexes that can be solubilized from the inner membrane using various detergents. A complex is then purified using a combination of other methods for detailed characterization of its components. Altogether, there are 69 different polypeptide chains. Complexes I, III, and IV are three large multisubunit proteins that span the inner membrane. All three function as proton pumps. They are the most complex enzymes known. Complex II does not function as a proton pump. 62

Figure 3. The mitochondrial electron transport chain. The number of protons transported across the membrane at each complex remains controversial. The best estimates indicate that 2 to 4 H + are ejected at each complex. Since oxygen and ATP are much easier to measure, the P/O ratio (ATP equivalents per oxygen atom) provides important information. These measurements yield values close to 2.5 for NADH and 1.5 for FADH 2. For convenience, however, the ATP yields are rounded off to 3 and 2, respectively. The lower P/O ratio for the oxidation of FADH 2 makes sense, since electrons from the flavin coenzyme enter the transport chain after Complex I. Consequently, the number of H + transported from the matrix per FADH 2 is lower. The ATP synthase requires the entry of 3 H + into the matrix per ATP synthesized. Coenzyme Q (CoQ), also known as ubiquinone, is lipophilic and highly mobile within the bilayer. It acts as an electron shuttle between complex I and III and between complex II and III. Cytochrome c is a water-soluble hemeprotein that carries electrons between complexes III and IV. Complex I (NADH dehydrogenase) catalyzes the oxidation of NADH by CoQ (ubiquinone) and is the entry point for electrons travelling from NADH to O 2 via the electron transport chain. Complex I is the most complex enzyme that has been characterized, since it contains 41 different protein subunits, seven of which are encoded by mitochondrial DNA. Complex II is the smallest of the complexes. It consists of only four proteins, including succinate dehydrogenase. This flavoprotein catalyzes the oxidation of succinate to fumarate, passing electrons to FAD and finally to coenzyme Q. None of the proteins in complex II are encoded by the mitochondrial genome. Complex III (cytochrome c reductase) catalyzes the oxidation of reduced coenzyme Q by cytochrome c. This complex has 11 subunits. The five largest are also found in the electron transfer chains of chloroplasts and are highly conserved. The five include cytochrome b, cytochrome C1, the Rieski iron sulfur protein, and two core proteins. Cytochrome b is the only subunit of complex III encoded by mitochondrial DNA. 63

Cytochrome c oxidase (complex IV) is a transmembrane protein composed of 13 subunits, including cytochromes a and a3. This complex catalyzes the oxidation of reduced cytochrome c. Three of the subunits of Complex IV are encoded in the mitochondrial genome. Cytochrome c oxidase catalyzes the one-electron oxidations of four consecutive reduced cytochrome c molecules and the concomitant four-electron reduction of one O 2 molecule. The best way to understand the respiratory chain is to think of each complex as a multistep catalyst: simply forget all the enzymes and electron carriers housed in each complex. That way you can write out the following conceptual scheme for the electron transport system: 2.4 The transduction of redox energy into adenylate energy involves electrochemical energy generated by the redox-driven extrusion of protons from the matrix. Energy released by electron transfer drives H + across the inner membrane, from the matrix (inside) to the intermembrane space (outside). For each H + ejected, one anionic charge remains behind. As more electrons flow down the chain of electron carriers, hydrogen ions accumulate on one side of the membrane, leaving the other side with a negative charge. Just like a battery, separation of charge can be used to do work. We can play a compact disc in our 'Walkman ' or turn on a flashlight by letting electrons in a battery flow back from the negative pole to the positive pole. Eventually the charge difference between the poles evens out and we have to recharge the battery. Cells allow hydrogen ions to flow back across the charged inner membrane through an enzyme that can use the energy to make ATP. Separation of charge across the inner membrane creates an electrical potential called Ψ. This potential is created by electron transport (electron flow from high to low free energy). We can now see that pumping protons across the inner membrane takes energy because H + ions are being forced to move against (1) a concentration gradient (low inside, high outside), and (2) an electrical gradient across the inner membrane (negative inside, positive outside). The total free energy associated with the translocation of one proton is where F is the Faraday and Ψ is electrical potential across the membrane in volts. Because ph = log[h + ], we can write equation 1 as 64

Equation 2 expresses both the electrical and chemical components of the work required to move n H + ions across the inner mitochondrial membrane. When protons are pumped against the electrochemical gradient, both terms on the right-hand side of this equation have positive values, and therefore G is positive. When protons flow down their electrochemical gradient, the same amount of free energy becomes available to do work. In other words, G is the proton motive force. Sometimes it is convenient to express the proton motive force as p, which is simply G divided by nf. Expressing Ψ in millivolts, the amounts to defining the proton motive force as a "voltage" and immediately informs us about the status of the proton circuit (in the same way that a voltmeter tells us whether our car battery is fully charged). Although we often refer to the proton electrochemical gradient as a ph gradient, this is an oversimplification. The energy stored in the "proton concentration gradient" has two components, electrical and chemical. In a typical liver mitochondrion, the proton motive is about 200 mv. The transmembrane potential is about 160 mv and a ph of 0.75 accounts for the rest: Thus, the amount of energy available from the electrical gradient ( Ψ) is actually greater than the potential energy stored in the chemical ( ph) gradient. However, there are situations where the ph term dominates. In any case, the energy available for ATP synthesis depends on p, so that the number of protons that must flow through the ATP synthase complex varies with p. The intact membrane-bound matrix compartment is absolutely necessary for oxidative phosphorylation. This fact, along with many other sound experimental observations, indicates that the proton motive force established across the inner membrane plays a central role in converting redox energy into adenylate energy. Uncouplers such as 2,4-dinitrophenol (DNP) prevent the synthesis of ATP but do not inhibit oxygen consumption or substrate oxidation. Uncouplers work by destroying the ph gradient. Dinitrophenol is lipophilic enough to cross the mitochondrial inner membrane by self-diffusion. This enables the phenol to ferry protons from a high to low concentration until the ph inside is the same as outside. No ph gradient means that none of the energy from the oxidation of NADH and FADH 2 is available to drive ATP synthesis by the F 0 F 1 - type proton-translocating adenosine triphosphatase (F 0 F 1 ATPase ). 65

ATP Synthases: One of Nature's Most Unique Machines 3.1 All ATP synthases have three distinct parts, giving them a "mushroom-like appearance. In animals, the F-type H + ATPases or ATP synthases are found in the inner mitochondrial membrane. In plants they are found both in the inner mitochondrial membrane and in the thylakoid membrane of chloroplasts. The bacterial F-type ATPase is found in the cytoplasmic membrane. The mitochondrial ATP synthase has 12 subunits, two of which are encoded in the mitochondrial genome. In most cells F-type ATPases usually function to make ATP. However, in anaerobic bacteria F-type ATPases do work in the direction of ATP hydrolysis to establish a proton motive force across the cytoplasmic membrane. Anaerobic bacteria then use the proton gradient to transport nutrients into the cell. All ATP synthases have three distinct parts, a headpiece or F l sector, a basepiece or F 0 sector, and a stalk connecting the headpiece and basepiece. The F 0 sector is a transmembrane protein; its role is to conduct energy stored in the electrochemical gradient to the F l sector outside the membrane. The role of the F l sector is to bind ADP and Pi and then use the gradient to drive the synthesis of ATP. Although ATP synthases vary in subunit complexity, the enzymes show considerable overall structural similarity and amino acid sequence homology, particularly between subunits most concerned with H + translocation and ATP synthesis. Thus we can be sure that the mechanism of action is the same in all organisms. A salient feature that has proven invaluable in biochemical experiments is that the F l and F 0 sectors can be separated and studied separately. Purified F l is water-soluble and catalyzes ATP hydrolysis; it contains three catalytic sites for ATP synthesis (or hydrolysis, if F l is detached from F 0 ). F 0 by itself carries out passive H + conduction, that is, it behaves as an "hole" or channel in the membrane. The channel allows H + ions to diffuse across the membrane from high to low H + concentration even if there is no membrane potential. The H + ATPase of Escherichia coli is a plasma membrane enzyme with two physiological roles. Under low p conditions (that is, under conditions where the scarcity of final electron acceptors limit electron flow through the electron transfer chain), the enzyme hydrolyzes cytosolic ATP and pumps protons into the periplasmic space, thus increasing p. Under conditions where electron transfer reactions can generate a large enough proton motive force across the plasma membrane, the enzyme catalyzes ATP synthesis. Most microbiologists, however, have used the name 'H + ATPase' for years and hesitate to call the enzyme by its other name, 'ATP synthase'. 3.2 ATP synthase can direct the synthesis of ATP whenever the proton motive force is large enough to force the release of tightly bound ATP. Mechanistically, the synthesis of ATP proceeds with inversion of configuration of the γ phosphorus atom. No covalent phosphorylated enzyme intermediate in involved, and the bridge O atom between the β and γ phosphorus atoms comes from ADP. These findings suggest a direct nucleophilic attack by ADP O on inorganic phosphate to yield ATP and H 2 O. Kinetic and thermodynamic studies suggest that the F 1 F 0 ATPase uses 2 H + to form an 66

ATP molecule. However, we do not fully understand the molecular derails of the energy transduction mechanism. The hydrolysis of ATP is reversible in the active site of ATP synthase. The synthesis of ATP in solution is very unfavorable, whereas the equilibrium constant for synthesis at the active site is close to unity (see Fig. 4). Two requirements must be met for the facile synthesis of ATP at the active site. First, the binding of ATP must be very strong. This pulls the equilibrium toward ATP synthesis. Second, the binding of ADP and Pi must be relatively weak, even though ADP and Pi contain almost the same chemical structures for binding to the enzyme that are found in ATP. This weak binding produces an unstable state (high free energy) of the bound ADP and Pi in the active site. The free energy change is near zero because there is very strong binding of ATP and very weak binding of ADP and Pi. The weak binding of ADP and Pi is the result of two factors: 1) A destabilization of bound ADP and Pi. This results from desolvation and unfavorable electrostatic interactions. Relief of these unfavorable interactions when ATP is synthesized favors ATP synthesis. (2) A decrease in the entropy of bound ADP and Pi at the active site, compared with ADP and Pi in solution. The most obvious, and possibly the most important, difference between the enzymic and nonenzymic synthesis of ATP in this system is that the enzymic reaction is intramolecular, whereas the nonenzymic reaction is bimolecular. Intramolecular reactions are more favorable than bimolecular reactions by factors of up to 10 8. Formation of a new covalent bond is improbable; it requires a large loss of entropy because of loss of translational and rotational freedom of the reactants. This loss is much smaller and the reaction is much more probable if the reactants are already tightly bound in exactly the correct position to react. A negative value of T S corresponds to a positive G, so that the loss of entropy favors ATP synthesis. 3.3 Whatever the mechanism of transducing redox energy into adenylate energy is, its actual working mode must comply with the needs of the cell. An increase in respiration occurs whenever ATP utilization increases. The increased rate of ATP hydrolysis causes a decrease in the cytosolic ATP/ADP ratio, which allows more rapid entry of H + through the F-type H + ATPase. The increased H + influx allows more rapid electron transfer along the respiratory chain, thereby increasing both H + efflux and oxygen consumption while preventing collapse of the proton gradient. At steady state, H + efflux equals H + influx, with about 10 6 H + zipping through each F 0 channel every second. Flow of H + in this circuit creates a proton current. The rate of respiration shows that cytochrome c oxidase is not working at full capacity. Regarding the mitochondrion as a black box, there are three entry ports: one for oxygen (diffusion across the inner membrane), transporters to bring fuel molecules and Pi inside, and a transport system to import ADP and export ATP. Slow entry at anyone entry port could keep the furnace from working at full capacity. 67

Figure 4. The hydrolysis of ATP is freely reversible in the active site of ATP synthase. In solution the synthesis of ATP is very unfavorable, whereas the equilibrium constant for synthesis at the active site is close to unity. Experiments have shown that the synthesis of ATP from bound ADP and Pi does not require energy from the electrochemical proton gradient. Rather, the free energy change for splitting out a water molecule is favorable because the reactants are no longer in an aqueous environment. ADP and Pi bind to the active site with a large decrease in translational and rotational entropy. Loss of solvation energies and the appearance of electrostatic repulsions between the bound ADP and Pi also contribute to the high free energy of the enzyme-adp-pi complex. In contrast, the enzyme binds ATP tightly, that is, the binding energy is fairly large, stabilizing the enzyme-atp complex. Therefore, the bound ADP and Pi can combine to form ATP in the absence of proton flow. However, the ATP formed in this way remains tightly bound to the enzyme. The flow of protons through the F 0 channel is thought to produce a conformational change in the F 1 head sufficient to release the ATP. In other words, energy from the proton gradient is necessary for the release of the newly synthesized ATP Control of Mitochondrial Respiration 4.1 Oxidative phosphorylation requires continuous transport of ADP and Pi across the mitochondrial inner membrane. Obviously oxidative phosphorylation requires provision of Pi and ADP in 1:1 stoichiometry. This requirement is met by the concomitant operation of two transporters in the mitochondrial inner membrane, the phosphate transporter and a unique adenine nucleotide translocator. Together, these two active transporters use about one-fourth of the proton current generated by the electron transport system. The Pi/H + symporter uses the proton chemical gradient ( ph) to bring Pi into the matrix, whereas adenine nucleotide translocator uses the electrical potential gradient ( Ψ) to import ADP into the matrix. Because ATP has four negative charges, whereas ADP has only three, the obligatory 1:1 exchange of mitochondrial ATP for cytosolic ADP via the adenine 68

nucleotide translocator moves one negative charge to the positive (cytosolic) side of the inner membrane. Obviously, ATP transfer out of, and ADP transfer into, the matrix would quickly discharge Ψ. However, the Pi/H + symporter moves one negative charge to the matrix side for each Pi imported. Therefore, ADP and Pi are made available to the ATP synthase without net flow of charge across the membrane. However, the provision of ADP and Pi depends on the perpetual flow of H + through the phosphate transporter. The active transport of ADP across the inner mitochondrial membrane is important because a low ADP concentration in the cytosol maximizes the free energy of ATP hydrolysis by maintaining a high ATP/ADP ratio in that metabolic compartment. By the same token, active transport of ADP into the matrix ensures a low ATP/ADP ratio at the site where ATP is synthesized, which lowers the free energy of ATP synthesis. 4.2 ADP phosphorylation in mitochondria is absolutely coupled to cellular respiration no electron flow occurs unless ADP and Pi are available in the matrix. ATP generation in mitochondria is absolutely coupled to cellular respiration. Electron flow through the electron transport system causes a build-up in the proton electrochemical potential. When the cost of pumping H + out of the matrix equals or exceeds the energy released by electron transfer from NADH to O 2, the proton pumping comes to a stop. Further electron flow through the electron transport system cannot continue unless the proton electrochemical potential across the mitochondrial inner membrane is discharged by the inward flux of protons. However, protons cannot flow through the F 0 sector of the F 0 F l ATPase unless ADP and Pi are available in the matrix. That is, proton flux through F 0 is regulated by the F l component. The rate of cellular respiration is determined by the rate of ATP hydrolysis in extra mitochondrial reactions. Cytochrome c oxidase activity depends on two factors: 1) the rate of electron supply from NADH via the respiratory chain, and 2) the rate of oxygen diffusion into the matrix compartment. Under most conditions the rate of oxygen diffusion is usually faster than the rate of ATP utilization, which in turn determines the size of the proton current. Hence the rate of oxygen consumption depends on the rate of A TP utilization. In summary, oxidative phosphorylation uses the energy released during the flow of electrons from NADH to O 2 to drive the reaction ADP + Pi A TP + H 2 O to the right. Tight coupling between respiratory electron transport and ATP generation minimizes the dissipation of chemical energy as heat. Without tight coupling between electron transport and ATP generation, all the redox energy would be dissipated as heat. The cell meets almost all its energy needs by converting ATP back to ADP and Pi. These products are immediately transported into the mitochondria and recycled back to ATP. Only with continued ATP utilization can electrons continue to flow to O 2. Thus, ATP utilization drives respiration. 69

SUMMARY The purpose of this chapter is to introduce the reader to the fascinating world of mitochondria and what they do. Cells use two general types of reactions to generate ATP. The first, substrate-level phosphorylation, occurs when a high-energy phosphate is transferred from a high-energy compound to ADP, yielding ATP. These reactions occur in the soluble phase and involve high-energy compounds formed during metabolic processes such as glycolysis. Much more important from a quantitative standpoint are oxidative phosphorylation and photosynthetic phosphorylation, where energy from a series of successive electron transfer reactions drives the translocation of protons across a biological membrane, thereby storing energy in the form of a proton electrochemical gradient. Subsequently, an ATP synthase provides a mechanism to "trap" this energy by synthesizing ATP from ADP and Pi. In eukaryotic cells, oxidative phosphorylation involves electron transfer enzymes located in the mitochondrial inner membrane. The oxidation of carbohydrates and fats to CO 2 and H 2 O produces most of the electrons for oxidative phosphorylation. Cells have evolved universal pathways (citric acid cycle and β oxidation) that, surprisingly, break down these fuels to CO 2 without using molecular oxygen. All the oxygen atoms in the CO 2 are derived from water or the fuel molecule itself. Nonetheless the fuel molecules are being oxidized, because electrons are being stripped off and transferred to an electron carrier as hydrogen or reducing equivalents (H = H + + e ). The major carrier of reducing equivalents is NADH, although another carrier, FADH 2, is also involved. These carriers or coenzymes deliver their high-energy electrons to the electron transport chain, initiating a series of electron transfer reactions from one carrier to the next in the chain. The carriers are alternately reduced and oxidized, therefore the term "redox" (reduction/oxidation) reactions. Ultimately, the electrons reach molecular oxygen, reducing it to water. Thus, high-energy electrons from metabolic fuels attain the lowest energy level for electrons in biological systems. The question is: what happens to the free energy released as the electrons flow "downhill"? As mentioned above, oxidative phosphorylation refers to the process by which mitochondria trap the free energy from redox reactions and use it to make ATP. The mechanism for transducing redox energy into adenylate energy involves storing part of the redox energy as a proton electrochemical gradient across the inner membrane. Proton pumps are located at three different locations along the electron transport chain. Electron transfer reactions at these sites pump protons across the inner membrane, resulting in the accumulation of protons on outside. The free energy stored in the proton gradient in turn drives the synthesis of ATP from ADP and Pi. To do so, the protons re-enter the matrix via the proton channel component of the enzyme ATP synthase, also known as F 0 F l ATPase or, simply, F-type ATPase. Tight coupling between respiratory electron transport and ATP generation minimizes the dissipation of chemical energy as heat. Protons cannot flow through the ATP synthase in the absence of ADP and Pi. During oxidative phosphorylation a Pi/H + symporter in the inner membrane transports Pi into the matrix. Another transport system, the adenine 70

nucleotide translocator, brings one ADP into the matrix while exporting an ATP molecule to the cytosolic compartment. Together, these two transport systems use about one-fourth of the proton current generated by the electron transport system. Under most physiologic conditions, the rate of electron transfer depends on the rate at which ADP is formed outside the mitochondria, that is, respiration is controlled by the cellular demand for ATP. This "respiratory control" ensures that ATP is never produced more rapidly than necessary. 71