Introduction

Having considered in general terms how mitochondria function, let us now look in more detail at the respiratory chain - the electron-transport chain that is so crucial to all oxidative metabolism. Most of the elements of the chain are intrinsic components of the inner mitochondrial membrane, and they provide some of the clearest examples of the many complicated interactions that can occur among the individual proteins located in a biological membrane.

Functional Inside-out Particles Can Be Isolated from Mitochondria ¹²

The respiratory chain is relatively inaccessible to experimental manipulation in intact mitochondria. By disrupting mitochondria with ultrasound, however, it is possible to isolate functional submitochondrial particles, which consist of broken cristae that have resealed into small closed vesicles about 100 nm in diameter (Figure 14-23). When these submitochondrial particles are examined in an electron microscope, their outside surfaces are seen to be studded with tiny spheres attached to the membrane by stalks (Figure 14-24). In intact mitochondria these lollipoplike structures are located on the inner (matrix) side of the inner membrane. Thus the submitochondrial particles are inside-out vesicles of inner membrane, with what was previously their matrix-facing surface exposed to the surrounding medium. As a result, they can readily be provided with the membrane-impermeable metabolites that would normally be present in the matrix space. When NADH, ADP, and inorganic phosphate are added, such preparations transport electrons from NADH to O₂ and couple this oxidation to ATP synthesis, catalyzing the reaction ADP + P_i→ ATP. This cell-free system provides an assay that makes it possible to purify the many proteins responsible for oxidative phosphorylation in a functional form.

Figure 14-23

Preparation of submitochondrial particles from purified mitochondria. The particles are pieces of broken-off cristae that form closed vesicles.

Figure 14-24

Electron micrograph of submitochondrial particles. This preparation has been negatively stained. (Courtesy of Efraim Racker.)

ATP Synthase Can Be Purified and Added Back to Membranes ¹³

The first experiments to show that the various membrane proteins that catalyze oxidative phosphorylation can be separated without destroying their activity were performed in 1960. The tiny protein spheres studding the surface of submitochondrial particles were stripped from the particles, leaving the stem of the lollipop and the other inner membrane proteins still in the particle membrane. The stripped particles could still oxidize NADH in the presence of oxygen, but they could no longer synthesize ATP. On the other hand, the purified spheres on their own acted as ATPases, hydrolyzing ATP to ADP and P_i. When the purified spheres (referred to as F₁ATPase) were added back to stripped submitochondrial particles, however, the reconstituted particles once again made ATP from ADP and P_i.

Subsequent work showed that the F₁ATPase is part of a larger transmembrane complex (~500,000 daltons) containing at least nine different polypeptide chains (Figure 14-25), which is now known as ATP synthase (also called F₀F₁ATPase). ATP synthase constitutes about 15% of the total inner membrane protein, and very similar enzyme complexes are present in both chloroplast and bacterial membranes. The transmembrane portion of the protein complex acts as a H⁺ carrier, and the F₁ATPase portion (the lollipop head) normally synthesizes ATP when protons pass through it down their electrochemical gradient. When separated from the H⁺ carrier, however, the F₁ATPase goes into reverse and catalyzes only ATP hydrolysis.

Figure 14-25

ATP synthase. As indicated, the F₁ATPase portion is formed from multiple subunits (Greek letters), as is the transmembrane H⁺ carrier.

One of the most convincing demonstrations of the function of ATP synthase came from an experiment performed in 1974. By that time methods had been developed for transferring detergent-solubilized integral membrane proteins into lipid vesicles (liposomes) formed from purified phospholipids. It thus became possible to form a hybrid membrane that contained both a complete purified mitochondrial ATP synthase and bacteriorhodopsin (a bacterial light-driven H⁺ pump, discussed in Chapter 10) but none of the proteins of the mitochondrial respiratory chain. When these vesicles were exposed to light, the H⁺ pumped into the vesicle lumen by the bacteriorhodopsin flowed back out through the ATP synthase, causing ATP to be made in the medium outside (Figure 14-26). Be-cause a direct interaction between a bacterial H⁺ pump and a mammalian ATP synthase seems highly unlikely, this experiment strongly suggests that in mitochondria the proton translocation driven by electron transport and the ATP synthesis are separate events.

Figure 14-26

An experiment demonstrating that the ATP synthase is driven by proton flow. By combining a light-driven bacterial proton pump (bacteriorhodopsin), an ATP synthase purified from ox heart mitochondria, and phospholipids, vesicles were produced that synthesized (more...)

ATP Synthase Can Function in Reverse to Hydrolyze ATP and Pump H^{+ 13}

ATP synthase can either use the energy of ATP hydrolysis to pump H⁺ across the inner mitochondrial membrane or it can harness the flow of H⁺ down an electrochemical proton gradient to make ATP (Figure 14-27). It thus acts as a reversible coupling device,interconverting electrochemical-proton-gradient and chemical-bond energies. Its direction of action depends on the balance between the steepness of the electrochemical proton gradient and the local Δ G for ATP hydrolysis.

Figure 14-27

ATP synthase is a reversible coupling device that interconverts the energies of the electrochemical proton gradient and chemical bonds. The ATP synthase can either synthesize ATP by harnessing the proton-motive force (top) or pump protons against their (more...)

The enzyme complex is called ATP synthase because it is normally driven by the large electrochemical proton gradient maintained by the respiratory chain (see Figure 14-20) to make most of the cell's ATP. The exact number of protons needed to make each ATP molecule is not known with certainty. To facilitate the calculations to be described below, however, we shall assume that one molecule of ATP is made by the ATP synthase for every three protons driven through it.

Whether the ATP synthase works in its ATP-synthesizing or its ATP-hydrolyzing direction at any instant depends on the exact balance between the favorable free-energy change for moving the three protons across the membrane into the matrix space (Δ G _3H⁺, which is less than zero) and the unfavorable free-energy change for ATP synthesis in the matrix (Δ G _{ATP synthesis}, which is greater than zero). As previously discussed, the value of Δ G _{ATP synthesis} depends on the exact concentrations of the three reactants ATP, ADP, and P_i in the mitochondrial matrix space (see Figure 14-22). The value of Δ G _3H⁺, on the other hand, is proportional to the value of the proton-motive force across the inner mitochondrial membrane. The following example will help to explain how the balance between these two free-energy changes affects the ATP synthase.

As explained in the legend to Figure 14-27, a single H⁺ moving into the matrix down an electrochemical gradient of 200 mV liberates 4.6 kcal/mole of free energy, while the movement of three protons liberates three times this much free energy (Δ G _3H⁺ = -13.8 kcal/mole). Thus, if the proton-motive force remains constant at 200 mV, the ATP synthase will synthesize ATP until a ratio of ATP to ADP and P_i is reached where Δ G _{ATP synthesis} is just equal to +13.8 kcal/mole (here Δ G _{ATP synthesis} + Δ G _3H⁺ = 0). At this point there will be no further net ATP synthesis or hydrolysis by the ATP synthase.

Suppose that a large amount of ATP is suddenly hydrolyzed by energy-requiring reactions in the cytosol - causing the ATP:ADP ratio in the matrix to fall. Now the value of Δ G _{ATP synthesis} will decrease (see Figure 14-22), and ATP synthase will begin to synthesize ATP again to restore the original ATP:ADP ratio. Alternatively, if the proton-motive force drops suddenly and is then maintained at a constant 160 mV, Δ G _3H⁺ will change to -11.0 kcal/mole. As a result, ATP synthase will start hydrolyzing some of the ATP in the matrix until a new balance of ATP to ADP and P_i is reached (where Δ G _{ATP synthesis} = +11.0 kcal/mole) and so on.

In many bacteria ATP synthase is routinely reversed in a transition between aerobic and anaerobic metabolism, as we shall see later. The reversibility of the ATP synthase is a property shared by other membrane proteins that couple ion movement to ATP synthesis or hydrolysis. Both the Na⁺-K⁺ pump and the Ca²⁺ pump described in Chapter 11, for example, hydrolyze ATP and use the energy released to pump specific ions across a membrane. If either of these pumps is exposed to an abnormally steep gradient of the ions it transports, however, it will act in reverse - synthesizing ATP from ADP and P_i instead of hydrolyzing it. Thus, like ATP synthase, such pumps are able to convert the electrochemical energy stored in a transmembrane ion gradient directly into phosphate bond energy in ATP.

The Respiratory Chain Pumps H⁺ Across the Inner Mitochondrial Membrane ¹⁴

The respiratory chain embedded in the inner mitochondrial membrane normally generates the electrochemical proton gradient that drives ATP synthesis. The ability of the respiratory chain to translocate H⁺ outward from the matrix space can be demonstrated experimentally under special conditions. A suspension of isolated mitochondria, for example, can be provided with a suitable substrate for oxidation, and the H⁺ flow through ATP synthase can be blocked. In the absence of air the injection of a small amount of oxygen into such a preparation causes a brief burst of respiration, which lasts for 1 to 2 seconds before all the oxygen is consumed. During this respiratory burst a sudden acidification of the medium resulting from the extrusion of H⁺ from the matrix space can be measured with a sensitive pH electrode.

In a similar experiment carried out with a suspension of submitochondrial particles, the medium becomes more basic when oxygen is injected, since H⁺ is pumped into each vesicle because of its inside-out orientation.

Spectroscopic Methods Have Been Used to Identify Many Electron Carriers in the Respiratory Chain ¹⁵

Many of the electron carriers in the respiratory chain absorb visible light and change color when they are oxidized or reduced. In general, each has an absorption spectrum and reactivity that is distinct enough to allow its behavior to be traced spectroscopically even in crude mixtures. It was therefore possible to purify these components long before their exact functions were known. Thus the cytochromes were discovered in 1925 as compounds that undergo rapid oxidation and reduction in living organisms as disparate as bacteria, yeasts, and insects. By observing cells and tissues with a spectroscope, three types of cytochromes were identified by their distinctive absorption spectra and designated cytochromes a, b, and c. This nomenclature has survived even though cells are now known to contain several cytochromes of each type and the classification into types is not functionally important.

The cytochromes constitute a family of colored proteins that are related by the presence of a bound heme group whose iron atom changes from the ferric (Fe III) to the ferrous (Fe II) state whenever it accepts an electron. The heme group consists of a porphyrin ring with a tightly bound iron atom held by four nitrogen atoms at the corners of a square (Figure 14-28). A related porphyrin ring is responsible for the red color of blood and the green color of leaves, being bound to iron in hemoglobin and to magnesium in chlorophyll. The best understood of the many proteins in the respiratory chain is cytochrome c, whose three-dimensional structure has been determined by x-ray crystallography (Figure 14-29).

Figure 14-28

The structure of the heme group attached covalently to cytochrome c. The porphyrin ring is shown in blue. There are five different cytochromes in the respiratory chain. Because the hemes in different cytochromes have slightly different structures and (more...)

Figure 14-29

The three-dimensional structure of cytochrome c, an electron carrier in the electron-transport chain. This small protein contains just over 100 amino acids and is held loosely on the membrane by ionic interactions (see Figure 14-33). The iron atom (orange (more...)

Iron-sulfur proteins are a second major family of electron carriers. In these proteins either two or four iron atoms are bound to an equal number of sulfur atoms and to cysteine side chains, forming an iron-sulfur center on the protein (Figure 14-30). There are more iron-sulfur centers than cytochromes in the respiratory chain, but their spectroscopic detection requires electron spin resonance (ESR) spectroscopy, and they are less well characterized.

Figure 14-30

The structures of two types of iron-sulfur centers. (A) A center of the 2Fe2S type. (B) A center of the 4Fe4S type. Although they contain multiple iron atoms, each iron-sulfur center can carry only one electron at a time. There are more than six different (more...)

The simplest of the electron carriers is a small hydrophobic molecule dissolved in the lipid bilayer known as ubiquinone, or coenzyme Q. A quinone (Q) can pick up or donate either one or two electrons, and it temporarily picks up a proton from the medium along with each electron that it carries (Figure 14-31).

Figure 14-31

Quinones. Each of these electron carriers in the respiratory chain picks up one H⁺ from the aqueous environment for every electron it accepts, and it can carry either one or two electrons as part of a hydrogen atom (yellow). When it donates its electrons (more...)

In addition to six different hemes linked to cytochromes, more than six iron-sulfur centers, and ubiquinone, there are also two copper atoms and a flavin serving as electron carriers tightly bound to respiratory-chain proteins in the pathway from NADH to oxygen. The pathway involves about 40 different proteins in all. The order of the individual electron carriers in the chain has been determined by sophisticated spectroscopic measurements (Figure 14-32), and many of the proteins were initially isolated and characterized as individual polypeptides. A major advance in understanding the respiratory chain, however, was the later realization that most of the proteins are organized into three large enzyme complexes.

Figure 14-32

The general methods used to determine the path of electrons along an electron-transport chain. The extent of oxidation of electron carriers a, b, c, and d is continuously monitored by following their distinct spectra, which differ in their oxidized and (more...)

The Respiratory Chain Contains Three Large Enzyme Complexes Embedded in the Inner Membrane ¹⁶

Membrane proteins are difficult to purify as intact complexes because they are insoluble in most aqueous solutions, and some of the detergents required to solubilize them can destroy normal protein-protein interactions. In the early 1960s, however, it was found that relatively mild ionic detergents, such as deoxycholate, will solubilize selected components of the mitochondrial inner membrane in their native form. This permitted the identification and purification of the three major membrane-bound respiratory enzyme complexes in the pathway from NADH to oxygen (Figure 14-33). As we shall see, each of these complexes acts as an electron-transport-driven H⁺ pump; they were initially characterized, however, in terms of the electron carriers that they interact with and contain.

Figure 14-33

The path of electrons through the three respiratory enzyme complexes. The size and shape of each complex is shown, as determined from images of two-dimensional crystals (crystalline sheets) viewed in the electron microscope at various tilt angles. During (more...)

1.

The NADH dehydrogenase complex is the largest of the respiratory enzyme complexes, with a mass of about 800,000 daltons and more than 22 polypeptide chains. It accepts electrons from NADH and passes them through a flavin and at least five iron-sulfur centers to ubiquinone, which transfers its electrons to a second respiratory enzyme complex, the b-c₁ complex.

2.

The cytochrome b-c₁ complex contains at least 8 different polypeptide chains and is thought to function as a dimer of about 500,000 daltons. Each monomer contains three hemes bound to cytochromes and an iron-sulfur protein. The complex accepts electrons from ubiquinone and passes them on to cytochrome c, which carries its electron to the cytochrome oxidase complex.

The cytochrome oxidase complex (cytochrome aa₃) is the best characterized of the three complexes. It is isolated as a dimer of about 300,000 daltons; each monomer contains at least 9 different polypeptide chains, including two cytochromes and two copper atoms. The complex accepts electrons from cytochrome c and passes them to oxygen.

The cytochromes, iron-sulfur centers, and copper atoms can carry only one electron at a time. Yet each NADH donates two electrons, and each O₂ molecule must receive four electrons to produce water. There are several electron-collecting and electron-dispersing points along the electron-transport chain where these changes in electron number are accommodated.

An Iron-Copper Center in Cytochrome Oxidase Catalyzes Efficient O₂ Reduction ¹⁷

Because oxygen has a high affinity for electrons, it releases a large amount of free energy when it is reduced to form water. Thus the evolution of cellular respiration, in which O₂ is converted to water, enabled organisms to harness much more energy than can be derived from anaerobic metabolism. This is presumably why all higher organisms respire. For biological systems to use O₂ in this way, however, requires a very sophisticated chemistry. We can tolerate O₂ in the air we breathe because it has trouble picking up its first electron, which allows its initial reaction in cells to be controlled closely by enzymatic catalysis. But once a molecule of O₂ has picked up one electron to form a superoxide radical (O₂ ^-), it becomes dangerously reactive and will rapidly take up an additional three electrons wherever it can find them. The cell can use O₂ for respiration only because cytochrome oxidase holds onto oxygen at a special bimetallic center (Figure 14-34B), where it remains clamped between a heme-linked iron atom and a copper atom until it has picked up a total of four electrons; only then can the two oxygen atoms of the oxygen molecule be safely released as two molecules of water (Figure 14-34C).

Figure 14-34

The reaction of O₂ with electrons in cytochrome oxidase. (A) The arrangement of electron carriers in cytochrome oxidase. Subunit I, which has 12 membrane-spanning alpha helices, contains two heme-linked iron atoms; one of these serves as an electron queuing (more...)

Although cytochrome oxidase contains many protein subunits, most of these appear to have a subsidiary role, helping to regulate either the activity or the assembly of the three subunits that form the core of the enzyme. One of the core subunits contains the bimetallic center where oxygen is bound, and it is responsible for pumping the four protons that are transferred across the inner mitochondrial membrane for each O₂ molecule that is reduced to water (see Figure 14-34).

The cytochrome oxidase reaction is estimated to account for 90% of the total oxygen uptake in most cells. Cyanide and azide are toxic to cells because they bind tightly to this complex and thereby block all electron transport.

Electron Transfers Are Mediated by Random Collisions Between Diffusing Donors and Acceptors in the Mitochondrial Inner Membrane ¹⁸

The two components that carry electrons between the three major enzyme complexes of the respiratory chain - ubiquinone and cytochrome c - diffuse rapidly in the plane of the inner mitochondrial membrane. The expected rate of random collisions between these mobile carriers and the enzyme complexes can account for the observed rates of electron transfer (each complex donates and receives an electron about once every 5 to 20 milliseconds). Thus there is no need to postulate a structurally ordered chain of electron-transfer proteins in the lipid bilayer; indeed, the three enzyme complexes appear to exist as independent entities in the plane of the inner membrane, and the ordered transfer of electrons is due entirely to the specificity of the functional interactions among the components of the chain.

This view is supported by the observation that the various components of the respiratory chain are present in different amounts. For each molecule of NADH dehydrogenase complex in heart mitochondria, for example, it is estimated that there are 3 molecules of b-c₁ complex, 7 molecules of cytochrome oxidase complex, 9 molecules of cytochrome c, and 50 molecules of ubiquinone; very different ratios are found in the mitochondria of some other cells. These components form a chain in the sense that each interacts specifically only with the carrier adjacent to it in the sequence shown in Figure 14-33, and there is a net flow of electrons from NADH dehydrogenase to cytochrome oxidase because each of the enzyme complexes in the sequence has a higher affinity for electrons than its predecessor. The affinity of a molecule for electrons is its redox potential. The changes in redox potential from one electron carrier to the next are exploited to pump proteins out of the mitochondrial matrix, as we now discuss.

A Large Drop in Redox Potential Across Each of the Three Respiratory Enzyme Complexes Provides the Energy for H⁺ Pumping ¹⁹

Pairs of compounds such as H₂O and 1/2 O₂, or NADH and NAD⁺, are called conjugate redox pairs, since one compound is converted to the other by adding one or more electrons plus one or more protons - the protons being readily available in any aqueous solution. Thus, for example,

Many readers will know that a 50-50 (equimolar) mixture of the members of a conjugate acid-base pair acts as a buffer, maintaining a defined "H⁺ pressure," or pH, which is a measure of the dissociation constant of the acid. In exactly the same way a 50-50 mixture of the members of a conjugate redox pair maintains a defined "electron pressure," or redox (reduction-oxidation) potential, E, that is a measure of the electron carrier's affinity for electrons.

By placing electrodes in contact with solutions that contain the appropriate conjugate redox pairs, one can measure the redox potential of each of the various electron carriers that participate in biological oxidation-reduction reactions. For biological systems each redox potential is determined at pH 7.0, where [H⁺] = 10^-7M. Those pairs of compounds that have the most negative redox potentials have the weakest affinity for electrons and therefore contain carriers with the strongest tendency to donate electrons, whereas pairs that have the most positive redox potentials have the strongest affinity for electrons and contain carriers with the strongest tendency to accept electrons. Thus a 50-50 mixture of NADH and NAD⁺ has a redox potential of -320 mV, indicating that NADH has a strong tendency to donate electrons; a 50-50 mixture of H₂O and 1/2 O₂ has a redox potential of +820 mV, indicating that O₂ has a strong tendency to accept electrons.

Redox potentials can be readily determined for all the electron carriers in the respiratory chain that can be distinguished by their spectra, and they can be shown to increase as one passes along the chain of electron carriers. As most cytochromes have higher redox potentials than iron-sulfur centers, they generally serve as electron carriers near the O₂ end of the respiratory chain, whereas the iron-sulfur proteins serve as carriers near the NADH end.

An outline of the redox potentials measured along the respiratory chain is shown in Figure 14-35. The potentials drop in three large steps, one across each major enzyme complex. The change in redox potential between any two electron carriers is directly proportional to the free energy released by an electron transfer between them (see Figure 14-35). Each complex acts as an energy-conversion device, harnessing this free-energy change to pump H⁺ across the inner membrane, thereby creating an electrochemical proton gradient as electrons pass through. This conversion can be demonstrated by incorporating each purified complex separately into liposomes: when an appropriate electron donor and acceptor is added so that electrons can pass through the complex, H⁺ is translocated across the liposome membrane.

Figure 14-35

The redox potential (denoted E'₀ or E _h) increases as electrons flow down the respiratory chain to oxygen. The standard free-energy change, Δ G°(in kilocalories per mole), for the transfer of the two electrons donated by an NADH molecule (more...)

The Mechanism of H⁺ Pumping Is Best Understood in Bacteriorhodopsin ²⁰

Because some respiratory enzyme complexes pump one H⁺ per electron across the inner mitochondrial membrane whereas others pump two, the molecular mechanism by which electron transport is coupled to H⁺ pumping is presumably different for the three different enzyme complexes. The details of the actual mechanisms are not known. In the case of the bc₁ complex, the quinones clearly play a part. As mentioned previously, a quinone picks up a H⁺ from the aqueous medium along with each electron it carries and liberates it when it releases the electron (see Figure 14-31). Since ubiquinone is freely mobile in the lipid bilayer, it could accept electrons near the inside surface of the membrane and donate them to the bc₁ complex near the outside surface, thereby transferring one H⁺ across the bilayer for every electron transported. Two protons are pumped per electron in the bc₁ complex, however, and there is evidence for a so-called Q-cycle, in which ubiquinone is recycled through the complex in an ordered way that makes this two-for-one transfer possible.

Allosteric changes in protein conformations driven by electron transport can also pump H⁺, just as H⁺ is pumped when ATP is hydrolyzed by the ATP synthase running in reverse. For both the NADH dehydrogenase complex and the cytochrome oxidase complex, it seems likely that electron transport drives orderly allosteric changes in protein conformation that cause a portion of the protein to pump H⁺ across the inner mitochondrial membrane. This type of proton pumping is best understood for bacteriorhodopsin, a light-driven H⁺ pump found in the plasma membrane of certain highly specialized bacteria (see Figure 10-32). A general mechanism for H⁺ pumping based on structural and functional studies of this protein is presented in Figure 14-36.

Figure 14-36

H⁺ pumping. This general model for energy-driven H⁺ pumping is based on the mechanism that is thought to be utilized by bacteriorhodopsin. The transmembrane protein shown is driven through a cycle of three conformations, denoted here as A, B, and C. In (more...)

H⁺ Ionophores Dissipate the H⁺ Gradient and Thereby Uncouple Electron Transport from ATP Synthesis ²¹

Since the 1940s several substances, such as 2,4-dinitrophenol, have been known to act as uncoupling agents, uncoupling electron transport from ATP synthesis. The addition of these low-molecular-weight organic compounds to cells stops ATP synthesis by mitochondria without blocking their uptake of oxygen. In the presence of an uncoupling agent electron transport and H⁺ pumping continue at a rapid rate, but no H⁺ gradient is generated. The explanation for this effect is both simple and elegant: uncoupling agents are lipid-soluble weak acids that act as H⁺ carriers (H⁺ ionophores) and provide a pathway in addition to the ATP synthase for the flow of H⁺ across the inner mitochondrial membrane. As a result of this "short-circuiting," the proton-motive force is dissipated completely, and ATP can no longer be made.

Respiratory Control Normally Restrains Electron Flow Through the Chain ²²

When an uncoupler such as dinitrophenol is added to cells, mitochondria increase their oxygen uptake substantially because of an increased rate of electron transport. This increase reflects the existence of respiratory control. The control is thought to act via a direct inhibitory influence of the electrochemical proton gradient on the rate of electron transport. When the gradient is collapsed by an uncoupler, electron transport is free to run unchecked at the maximal rate. As the gradient increases, electron transport becomes more difficult and the process slows. Moreover, if an artificially large electrochemical proton gradient is experimentally created across the inner membrane, normal electron transport stops completely and a reverse electron flow can be detected in some sections of the respiratory chain. This observation suggests that respiratory control reflects a simple balance between the free-energy change for electron-transport-linked proton pumping and the free-energy change for electron transport - that is, the magnitude of the electrochemical proton gradient affects both the rate and the direction of electron transport, just as it affects the directionality of the ATP synthase (see Figure 14-27).

Respiratory control is just one part of an elaborate interlocking system of feedback controls that coordinates the rates of glycolysis, fatty acid breakdown, the citric acid cycle, and electron transport. The rates of all of these processes are adjusted to the ATP:ADP ratio, increasing whenever increased utilization of ATP causes the ratio to fall. The ATP synthase in the inner mitochondrial membrane, for example, works faster as the concentrations of its substrates ADP and P_i increase. As it speeds up, the enzyme lets more H⁺ flow into the matrix and thereby dissipates the electrochemical proton gradient more rapidly. The falling gradient, in turn, enhances the rate of electron transport.

Similar controls, including feedback inhibition of several key enzymes by ATP (see Figure 14-13, for example), act to adjust the rates of NADH production to the rate of NADH utilization by the respiratory chain and so on. As a result of these many control mechanisms, the body oxidizes fats and sugars 5 to 10 times more rapidly during a period of strenuous exercise than during a period of rest.

Natural Uncouplers Convert the Mitochondria in Brown Fat into Heat-generating Machines ²³

In some specialized fat cells mitochondrial respiration is normally uncoupled from ATP synthesis. In these cells, known as brown fat cells, most of the energy of oxidation is dissipated as heat rather than being converted into ATP. The inner membranes of the large mitochondria in these cells contain a special transport protein that allows protons to move down their electrochemical gradient without activating ATP synthase. As a result, the cells oxidize their fat stores at a rapid rate and produce more heat than ATP. Tissues containing brown fat thereby serve as "heating pads" that revive hibernating animals and protect sensitive areas of newborn human babies from the cold.

All Bacteria Use Chemiosmotic Mechanisms to Harness Energy ²⁴

Bacteria use enormously diverse energy sources. Some, like animal cells, are aerobic and synthesize ATP from sugars that they oxidize to CO₂ and H₂O by glycolysis and the citric acid cycle through a respiratory chain in their plasma membrane similar to that in the mitochondrial inner membrane. Others are strict anaerobes, deriving their energy either from glycolysis alone (by fermentation) or, in addition, from an electron-transport chain that employs a molecule other than oxygen as the final electron acceptor. The alternative electron acceptor can be a nitrogen compound (nitrate or nitrite), a sulfur compound (sulfate or sulfite), or a carbon compound (fumarate or carbonate), for example. The electrons are transferred to these acceptors by a series of electron carriers in the plasma membrane that are comparable to those in mitochondrial respiratory chains.

Despite this diversity, the plasma membrane of the vast majority of bacteria contains an ATP synthase that is very similar to that in mitochondria (and chloroplasts). In aerobic bacteria the electron-transport chain pumps H⁺ out of the cell and thereby establishes a proton-motive force that drives the ATP synthase to make ATP. In anaerobic bacteria that lack an electron-transport chain, the ATP synthase works in reverse, using the ATP produced by glycolysis to pump H⁺ and establish a proton-motive force across the bacterial plasma membrane.

Thus most bacteria, including the strict anaerobes, maintain a proton-motive force across their plasma membrane. It can be harnessed to drive a flagellar motor that enables the bacterium to swim and is used to pump Na⁺ out of the bacterium via a Na⁺-H⁺ antiporter that takes the place of the Na⁺-K⁺ ATPase of eucaryotic cells. It is also used for the active transport of nutrients, such as most amino acids and many sugars, into bacteria: each nutrient is dragged into the cell along with one or more H⁺ through a specific symporter (Figure 14-37). In animal cells, by contrast, most inward transport across the plasma membrane is driven by the Na⁺ gradient established by the Na⁺-K⁺ ATPase.

Figure 14-37

H⁺-driven transport in bacteria. A proton-motive force generated across the plasma membrane pumps nutrients into the cell and expels sodium. In (A) the electrochemical proton gradient is generated in an aerobic bacterium by a respiratory chain and is (more...)

Some unusual bacteria have adapted to live in a very alkaline environment and yet must maintain their cytoplasm at a physiological pH. For these cells any attempt to generate an electrochemical H⁺ gradient would be opposed by a large H⁺ concentration gradient in the wrong direction (H⁺ higher inside than outside). Presumably for this reason, at least some of these bacteria substitute Na⁺ for H⁺ in all of their chemiosmotic mechanisms. The respiratory chain pumps Na⁺ out of the cell, the transport systems and flagellar motor are driven by an inward flux of Na⁺, and a Na⁺-driven ATP synthase synthesizes ATP. The existence of such bacteria demonstrates that the principle of chemiosmosis is more fundamental than the proton-motive force on which it is normally based.

Summary

The respiratory chain in the inner mitochondrial membrane contains three major enzyme complexes through which electrons pass on their way from NADH to O₂. Each of these can be purified, inserted into synthetic lipid vesicles, and then shown to pump H⁺ when electrons are transported through it. In the native membrane the mobile electron carriers ubiquinone and cytochrome c complete the electron-transport chain by shuttling between the enzyme complexes. The path of electron flow is NADH → NADH dehydrogenase complex → ubiquinone → b-c₁ complex → cytochrome c → cytochrome oxidase complex → molecular oxygen (O₂).

The respiratory enzyme complexes couple the energetically favorable transport of electrons to the pumping of H⁺ out of the matrix. The resulting electrochemical proton gradient is harnessed to make ATP by another transmembrane protein complex, ATP synthase, through which H⁺ flows back into the matrix. The ATP synthase is a reversible coupling device that normally converts a backflow of H⁺ into ATP phosphate-bond energy by catalyzing the reaction ADP + P_i → ATP, but it can also work in the opposite direction and hydrolyze ATP to pump H⁺ if the electrochemical proton gradient is reduced. Its universal presence in mitochondria, chloroplasts, and bacteria testifies to the central importance of chemiosmotic mechanisms in cells.