Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Molecular Organization of Cells
Energy Conversion: Mitochondria and Chloroplasts
Respiratory Chain and ATP Synthase

Having outlined the general function of Cell/35.html">Mitochondria, we now turn to a more detailed examination of the Respiratory Chain—the Electron Transport Chain, which is of paramount importance for oxidative METABOLISM as a whole. Most components of this chain are an integral part of The inner mitochondrial membrane and serve as one of the most striking Examples of complex interactions between individual Proteins within a biological membrane.

7.2.1. Functionally active "inside-out" submitochondrial particles can be isolated from mitochondria [13]

In intact mitochondria, the respiratory chain is relatively inaccessible to experimental analysis. However, by subjecting mitochondria to ultrasound, one can obtain functionally active submitochondrial particles—fragments of cristae that have sealed into small vesicles about 100 nm in diameter (Fig. 7-23). When using negative staining, Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF submitochondrial particles reveal that their outer surface is studded with tiny spheres attached to the membrane by a "stalk" (Fig. 7-24). In intact mitochondria, these mushroom-like structures are located on the inner (matrix-facing) side of the inner membrane. Thus, submitochondrial particles represent inside-out fragments of the inner membrane: their surfaces, which previously faced the matrix, are now exposed to the surrounding medium. As a result, they are easily accessible to membrane-impermeant substances that are normally confined to the matrix. Upon addition of NADH, ADP, and inorganic phosphate, these particles transport electrons from NADH to O2, coupling this oxidation to ATP synthesis. Such a cell-free system makes it possible to isolate, in functionally active form, the numerous proteins responsible for Oxidative Phosphorylation.

7.2.2. ATP synthase can be isolated and reconstituted into the membrane in an active form [14]

In 1960, it was first demonstrated that various Membrane Proteins involved in oxidative phosphorylation could be isolated without losing their activity. The tiny protein structures studding The surface of submitochondrial particles were successfully detached and solubilized. Although submitochondrial particles depleted of these spherical structures continued to oxidize NADH in the presence of oxygen, no ATP synthesis occurred. On the other hand, the isolated structures acted as ATPases, hydrolyzing ATP to ADP and P_i. When these spherical structures (called F₁-ATPases) were added back to the depleted submitochondrial particles, the reconstituted particles once again synthesized ATP from ADP and P_i.

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Fig. 7-23. Preparation of submitochondrial particles from mitochondria. The particles are fragments of disrupted cristae that have sealed to form closed vesicles.

Fig. 7-24. Electron micrograph of submitochondrial particles. (Courtesy of Efraim Racker.)

It was subsequently shown that the F₁-ATPase is part of a large, membrane-spanning complex (with a molecular mass of about 500,000 daltons) consisting of at least nine different polypeptide chains. This complex is called ATP synthase (or F₀F₁-ATPase); it constitutes about 15% of the total protein of the inner mitochondrial membrane. Very similar ATP synthases are found in chloroplast and bacterial membranes. This protein complex contains transmembrane proton channels, and ATP synthesis occurs only when protons pass through these channels down their electrochemical gradient.

Grid-mapping experiments conducted in 1974 elegantly demonstrated how ATP synthase Functions. By that time, Methods had already been developed for reconstituting integral membrane proteins, previously solubilized with detergent, into lipid vesicles (Liposomes) prepared from purified Phospholipids (see Sec. 6.1.2). This allowed the creation of a "hybrid" membrane containing both purified mitochondrial ATP synthase and Bacteriorhodopsin, which functions as a light-driven proton pump in Bacteria (see Sec. 6.2.7). When these vesicles were illuminated, protons pumped inside by bacteriorhodopsin flowed back out through the ATP synthase, resulting in the accumulation of ATP in the surrounding solution (Fig. 7-25). Since a direct interaction between a bacterial proton pump and a mammalian ATP synthase is highly unlikely, this experiment indicates that in mitochondria, active proton transport and ATP synthesis are also, in all probability, two separate processes in the overall Mechanism of Oxidative phosphorylation.

7.2.3. ATP synthase can run in reverse, hydrolyzing ATP and pumping protons [16]

The action of ATP synthase is reversible: it can use either the energy of ATP Hydrolysis to pump protons across the inner mitochondrial membrane, or the energy of proton flow down an electrochemical gradient to synthesize ATP (Fig. 7-26). Thus, ATP synthase is a reversible coupling device that interconverts the energy of an electrochemical proton gradient and chemical bonds. The direction of its operation depends on the balance between the steepness of the proton gradient and the local ∆G for ATP hydrolysis.

ATP synthase is so named because, under normal conditions of a proton gradient maintained by the respiratory chain (see Fig. 7-20), it synthesizes most of The Cell's ATP. The exact number of protons required to synthesize one ATP molecule is not known with certainty. To simplify the calculations below, we will assume that for every three protons passing through the ATP synthase, one ATP molecule is synthesized.

Fig. 7-25. Diagram of a landmark experiment demonstrating that ATP synthase can be driven simply by a flow of protons. By combining a light-driven bacterial proton pump (bacteriorhodopsin), ATP synthase isolated from beef Heart mitochondria, and phospholipids, liposomes were reconstituted that synthesize ATP when exposed to light.

Fig. 7-26. ATP synthase is a reversible coupling device for interconverting the energy of an electrochemical proton gradient and chemical bond energy. It is also known as F₀F₁-ATPase and consists of at least nine different polypeptide chains. Five of these chains form the spherical HEAD of the complex, called the F₁-ATPase. ATP synthase can either synthesize ATP using the energy of the proton-motive force (top) or pump protons against their electrochemical gradient at the expense of ATP hydrolysis (bottom).

As explained in the text, the direction of the enzyme's action at any given moment depends on the net free-energy change for the coupled processes—the movement of protons across the membrane and the synthesis of ATP from ADP and P_i.

We have previously shown that the Free energy of ATP hydrolysis depends on the concentrations of the three reactants—ATP, ADP, and P_i (see Fig. 7-22). The ∆G for ATP synthesis is the same value with the opposite sign (negative). The free energy of moving protons across the membrane is the sum of (1) the ∆G for moving one mole of any ion between regions with a potential difference ∆V and (2) the ∆G for moving a mole of any molecule between regions of different concentration. The equation for the proton-motive force given in Sec. 7.1.7 combines these same components, except that the concentration difference is replaced by its equivalent increment in Membrane Potential, yielding an expression for the "Electrochemical Potential" of the proton. Thus, the ∆G for proton movement and the proton-motive force reflect the same potential, but the former is measured in kilocalories and the latter in millivolts. The conversion factor between these units is the Faraday constant. Therefore, ∆GH⁺ = -0.023 (proton-motive force), where ∆GH⁺ is expressed in kilocalories per mole (kcal/mole) and the proton-motive force in millivolts (mV). If the electrochemical proton gradient is 220 mV, then ∆GH⁺ = 5.06 kcal/mole.

Whether ATP synthase operates at any given moment in the direction of ATP synthesis or hydrolysis depends on the precise balance between the free-energy changes for the passage of three protons across the membrane into the matrix (∆G₃H⁺ < 0) and for ATP Synthesis in the matrix (∆G_{ATP synthesis} > 0). As mentioned, the value of ∆G_{ATP synthesis} is determined by the concentrations of the three reactants in the mitochondrial matrix—ATP, ADP, and P_i (see Fig. 7-22). On the other hand, the value of ∆G₃H⁺ will be proportional to the proton-motive force across the inner membrane. The following example illustrates how the relationship between these two free-energy changes affects the operation of ATP synthase.

As explained in the legend to Fig. 7-26, The entry of one proton into the matrix down an electrochemical gradient of 220 mV releases 5.06 kcal/mole, and the entry of three protons releases three times as much energy (∆G3H+ = -15.2 kcal/mole). Thus, at a constant proton-motive force (220 mV), ATP synthase will continue to synthesize ATP until The ratio of ATP to ADP and P_i reaches a value at which ∆G_{ATP synthesis} is exactly equal to +15.2 kcal/mole (where ∆G_{ATP synthesis} + ∆G₃H⁺ = 0). Under these conditions, ATP synthesis will be precisely balanced by its hydrolysis.

Suppose that, due to energy-requiring reactions, a large amount of ATP is suddenly hydrolyzed in the Cytosol, leading to a drop in the ATP:ADP ratio in the mitochondrial matrix. In this case, ∆G_{ATP synthesis} will decrease (see Fig. 7-22), and ATP synthase will switch back to ATP synthesis until the original ATP:ADP ratio is restored. If, however, the proton-motive force suddenly drops and is maintained at a constant level of 200 mV, ∆G₃H⁺ will decrease to -13.8 kcal/mole. As a result, ATP synthase will begin to hydrolyze ATP, and this reaction will continue until the ratio of ATP to ADP concentrations reaches some new value (at which ∆G_{ATP synthesis} is +13.8 kcal/mole), and so on.

As we shall see later, in many bacteria, ATP synthase reverses its direction of action with every transition between aerobic and Anaerobic Metabolism. A similar reversibility is characteristic of other membrane Enzymes that couple ion transport to the synthesis or hydrolysis of ATP. For example, the sodium-potassium and calcium pumps (the latter is described in Chapter 6) hydrolyze ATP and use the released energy to pump specific ions across the membrane (see Sec. 6.4.5). If any of these pumps are forced to operate against an unusually steep gradient of the transported ions, they will run in reverse—synthesizing ATP from ADP and P_i instead of hydrolyzing ATP. Thus, like ATP synthase, these pumps are capable of converting the energy stored in a transmembrane ion gradient directly into the energy of ATP phosphate bonds.

7.2.4. The respiratory chain pumps H⁺ ions across the inner mitochondrial membrane [16]

If ATP synthase normally does not transport H⁺ out of the matrix, the respiratory chain located in the inner mitochondrial membrane under normal conditions pumps protons across this membrane, thereby generating an electrochemical proton gradient that drives ATP synthesis. Under certain conditions, the ability of the respiratory chain to pump protons out of the matrix can be demonstrated experimentally. For example, a suspension of isolated mitochondria can be supplied with a suitable substrate for oxidation while blocking proton flow through ATP synthase. Under anaerobic conditions, a small addition of oxygen to such a preparation triggers a burst of respiratory activity lasting one to two seconds—until all the oxygen is consumed. During this respiratory burst, a sudden acidification of the medium resulting from the expulsion of H⁺ ions from the mitochondrial matrix can be recorded using a sensitive pH electrode.

A similar experiment can be performed with a suspension of submitochondrial particles. In this case, when oxygen is bubbled through, the medium becomes alkaline because the membrane is "inside-out," causing protons to be pumped into the interior of the particles.

7.2.5. Many electron carriers can be identified using spectroscopic methods [17].

Most electron carriers that make up the respiratory chain absorb light, and their oxidation or reduction is accompanied by a color change. Typically, the absorption spectrum and reactivity of each carrier are sufficiently characteristic to allow changes in its state to be monitored by spectroscopy even in crude extracts. This made it possible to isolate such carriers long before their true function was understood. For example, Cytochromes were discovered in 1925 as compounds that are rapidly oxidized and reduced in organisms as diverse as Yeast, bacteria, and insects. By observing Cells and Tissues with a spectroscope, researchers were able to identify Three types of cytochromes that differed in their Absorption Spectra and were named cytochromes a, b, and c. This nomenclature has survived to this day, although it is now known that cells contain several species of cytochromes of each type, and this Classification does not reflect their function.

Cytochromes form a family of colored proteins united by the presence of a bound heme group in their molecule; upon accepting a single electron, the iron atom within the heme is reduced, transitioning from the Fe III to the Fe II state. Heme contains a porphyrin ring and an iron atom firmly bound by four nitrogen atoms located at the corners of a square (Fig. 7-27). Closely related porphyrin rings determine the red color of Blood and the green color of leaves by binding iron in Hemoglobin (sec. 10.5.3) and magnesium in chlorophyll (sec. 7.3.6). Among the many Proteins of the respiratory chain, cytochrome c is the best studied; its three-dimensional Structure was determined by X-ray crystallography (Fig. 7-28).

Iron-sulfur proteins form a second important group of electron carriers. In the molecules of these proteins, two or four iron atoms are bound to an equal number of sulfur atoms and to Cysteine side chains, forming the iron-sulfur center of the protein (Fig. 7-29). Iron-sulfur centers are more abundant in the respiratory chain than cytochromes, but their detection requires electron spin Resonance (ESR) spectroscopy, making iron-sulfur centers less well studied.

The simplest electron carrier is a small hydrophobic molecule dissolved in The Lipid Bilayer, known as ubiquinone or coenzyme Q. It is capable of accepting or donating either one or two electrons and temporarily captures a proton from the medium during The transfer of each electron (Fig. 7-30).

Fig. 7-27. Structure of heme covalently bound to cytochrome c. Four of the six coordination positions of iron are occupied by the porphyrin ring. The fifth and sixth coordination positions are oriented perpendicular to the plane of the ring. In almost all cytochromes, these two positions are occupied by amino acid side chains, which prevents other ligands from binding here. An exception is cytochrome a₃; just as in hemoglobin and Myoglobin, the sixth coordination position of iron in this component of cytochrome oxidase is free and can therefore bind oxygen.

There are five different cytochromes in the respiratory chain. Because the Hemes incorporated into different cytochromes differ somewhat in structure and are bound to their respective proteins in non-identical ways, their affinities for electrons also vary.

Fig. 7-28. Three-dimensional model of cytochrome c, one of the electron carriers in the respiratory chain. This small protein, containing slightly more than 100 amino acid residues, can move within the membrane because it is held only by ionic interactions (see Fig. 7-35). The iron atom (highlighted in darker tone) within the bound heme (lighter shading) is capable of transferring a single electron (see also Fig. 352, A).

Fig. 7-29. Structure of Two Types of iron-sulfur centers. A. A 2Fe2S type center. B. A 4Fe4S type center. Although the center contains multiple iron atoms, each center is capable of transferring only one electron at a time. The respiratory chain contains more than six different iron-sulfur centers.

In addition to the six different hemes in cytochrome molecules, more than six iron-sulfur centers, and ubiquinone, There are two copper atoms and a flavin serving as electron carriers, tightly bound to respiratory chain proteins all the way from NADH to oxygen. The electron transport pathway involves a total of about 40 different proteins. The linear arrangement of the various carriers in the respiratory chain was determined using complex spectroscopic measurements (Fig. 7-31), and initially many of these proteins were isolated and described as separate Polypeptides. However, a true understanding of respiratory chain function came later when it was discovered that the proteins are organized into three large enzyme complexes.

7.2.6. The respiratory chain contains three large enzyme complexes embedded in the inner membrane [18]

Membrane proteins are difficult to isolate as intact complexes because they are insoluble in most aqueous solutions, and substances such as detergents and urea, which are required for their solubilization, can disrupt normal Protein-Protein Interactions (sec. 6.2.2). However, in the early 1960s, it was discovered that relatively mild ionic detergents, such as deoxycholate (Fig. 7-32), can solubilize some Components of the mitochondrial inner membrane in their native form. This made it possible to identify and isolate the three major membrane-bound respiratory enzyme complexes along the pathway from NADH to oxygen (Fig. 7-33).

1. The NADH dehydrogenase complex is the largest of the respiratory enzyme complexes—it has a molecular weight exceeding 800,000 and contains more than 22 polypeptide chains. It accepts electrons from NADH and transfers them via flavin and at least five iron-sulfur centers to ubiquinone, a small lipid-soluble molecule (see Fig. 7-30) that delivers electrons to the second respiratory enzyme complex, the b-c₁ complex.

2. The b-c₁ complex consists of at least 8 different polypeptide chains and presumably exists as a dimer with a Molecular Weight of 500,000. Each monomer contains three heme groups bound to cytochromes and an iron-sulfur protein. The complex accepts electrons from ubiquinone and transfers them to cytochrome c, a small peripheral membrane protein (see Fig. 7-28), which subsequently delivers them to the cytochrome oxidase complex.

3. The cytochrome oxidase complex (cytochrome aa3) is the most thoroughly studied of the three complexes. It consists of at least eight different polypeptide chains and has been isolated as a dimer with a molecular weight of 300,000; each monomer contains two cytochromes and two copper atoms. This complex accepts electrons from cytochrome c and transfers them to oxygen.

Fig. 7-30. Quinones as key electron carriers in the respiratory chain. For each accepted electron, the quinone captures one proton from the surrounding aqueous environment; thus, it is capable of transferring either one or two electrons. When the quinone passes its electrons to the next carrier, the protons are released. In mammalian mitochondria, the quinone is ubiquinone (coenzyme Q), shown in the figure; the long hydrophobic tail that anchors ubiquinone in the membrane typically consists of 10 five-carbon isoprene units. In plants, the corresponding carrier is plastoquinon, which is very similar to ubiquinone. For simplicity, ubiquinone and plastoquinone are commonly referred to simply as quinones and designated as Q.

Cytochromes, iron-sulfur centers, and copper atoms can transfer only a single electron at a time. Meanwhile, each NADH molecule yields two electrons, and each O₂ molecule must accept four electrons during The formation of Water molecules. The electron transport chain features several electron-collecting and electron-distributing sites where discrepancies in electron numbers are reconciled. For instance, the cytochrome oxidase complex accepts four electrons one by one from cytochrome c molecules and ultimately transfers them to a single bound O₂ molecule, leading to the formation of two water molecules. At intermediate steps of this process, two electrons pass into the heme of cytochrome a and the protein-bound copper atom, Cu_A, before reaching the oxygen-binding site. In turn, the oxygen-binding site contains another copper atom and the heme of cytochrome a₃. However, the exact mechanism by which two water molecules are formed through the Interaction of a bound O₂ molecule with four protons remains incompletely understood.

In most cells, about 90% of all consumed oxygen interacts with cytochrome oxidase. The toxicity of poisons such as cyanide and azide stems from their ability to bind tightly to the cytochrome oxidase complex, thereby blocking all electron transport.

7.2.7. Electron transfer occurs via random collisions between electron Donors and acceptors diffusing within the inner mitochondrial membrane [19]

Two components that shuttle electrons between the three major enzyme complexes of the respiratory chain—ubiquinone and cytochrome c—move rapidly by diffusion within the plane of the membrane.

Fig. 7-31. General scheme of the method used to determine The pathway of electron transfer along the respiratory chain. The oxidation states of electron carriers a, b, c, and d are continuously monitored by changes in their spectra in the oxidized and reduced states. A. Under normal conditions, all carriers are partially oxidized. If a specific electron transport inhibitor is added, all carriers "downstream" of the inhibition site become more oxidized, whereas those "upstream" become more reduced. B. In the absence of oxygen, all carriers are in a fully reduced state. Upon the sudden Introduction of oxygen, the carriers transition to a partially oxidized form; this oxidation process exhibits a lag phase that is greater the further "upstream" the carrier is located in the chain.

Fig. 7-32. Molecular structures of the relatively mild anionic detergents cholate and deoxycholate. Although both detergents are potent enough to solubilize membrane proteins, they can frequently be used without compromising enzyme activity.

Fig. 7-33. Relative Sizes and Shapes of the three respiratory enzyme complexes. These crude three-dimensional models were constructed from images of two-dimensional crystals (crystalline sheets) observed at various angles using an Electron microscope.

Collisions between these mobile carriers and the enzyme complexes can readily account for the observed rates of electron transfer (each complex releasing and accepting one electron every 5–20 milliseconds). Consequently, there is no need to assume a rigid structural ordering of the carrier protein chain within the lipid bilayer; indeed, the enzyme complexes appear to exist within the membrane as independent entities, and ordered electron transfer is ensured solely by the Specificity of functional interactions among the chain components.

This view is further supported by the fact that the various components of the respiratory chain are present in vastly different proportions. For instance, in heart mitochondria, for each molecule of the NADH dehydrogenase complex there are 3 molecules of the b-c₁ complex, 7 molecules of the cytochrome oxidase complex, 9 molecules of cytochrome c, and 50 molecules of ubiquinone; quite distinct stoichiometric ratios of these proteins have also been found in certain other cell types.

7.2.8. A significant drop in redox potential across each of the three respiratory chain complexes provides the energy required for proton pumping [12, 20]

A pair such as H₂O and ¹/₂O₂ (or NADH and NAD⁺) is referred to as a conjugate redox pair, since one of its members is converted into the other upon The addition of one or more electrons and one or more protons (the latter are always abundantly available in any aqueous solution). For example,

¹/₂О₂ + 2е^- + 2Н⁺→ Н₂О.

It is well established that a 50:50 mixture of compounds forming a conjugate acid-base pair acts as a buffer that maintains a specific "proton pressure" (pH), the value of which is determined by the acid dissociation constant. In the exact same manner, a 50:50 mixture of the components of a conjugate redox pair maintains a specific "electron pressure," or redox potential E, which serves as a measure of the carrier molecule's affinity for electrons.

By placing electrodes into a solution containing appropriate redox pairs, one can measure the Redox Potential of each electron carrier involved in Biological Oxidation-reduction reactions. Pairs of compounds with the most negative redox potentials have the lowest electron affinity, meaning they contain carriers with the least tendency to accept electrons and the greatest tendency to donate them. For example, a 50:50 mixture of NADH and NAD⁺ has a redox potential of -320 mV, indicating a strong propensity of NADH to donate electrons, whereas the redox potential of an equimolar mixture of H₂O and ¹/₂O₂ is +820 mV, reflecting O₂'s strong tendency to accept electrons.

Redox potentials can be measured for all electron transport chain carriers that are spectrally distinct. For instance, one can bypass PARTS OF THE chain by adding small molecules that readily donate and accept electrons. Spectroscopic measurements are then performed to determine the ratio of oxidized to reduced forms for each carrier as the redox potential of the shunting solution is altered in a stepwise fashion. As expected, potentials were found to increase progressively along the chain of carriers. For most cytochromes, potentials are higher than those of iron-sulfur centers; accordingly, cytochromes are typically located near the O₂ end of the respiratory chain, whereas iron-sulfur proteins reside near the NADH end.

Fig. 7-34. Increase in redox potential (designated as E'₀ or E_h) as electrons travel down the respiratory chain to oxygen. The right-hand ordinate shows the Standard Free Energy change for the transfer of two electrons delivered by a single NADH molecule [∆G = - n (0.023)∆Е₀', where n is the number of electrons transferred across a redox potential span of ∆E₀' mV]. Within each respiratory enzyme complex, electrons pass sequentially through four or more individual carriers. As noted previously, a portion of the released energy is utilized by each enzyme complex to pump protons across the inner mitochondrial membrane. The exact number of protons pumped per transferred electron (n) is not precisely known. Two electrons derived from FADH₂—generated during Fatty acid oxidation (see Fig. 7-11) and in The Citric Acid Cycle (see Fig. 7-14)—yield less useful energy than two electrons from NADH. Because the redox potential of FADH₂ is close to 0 mV, the transfer of electrons from FADH₂ to ubiquinone is not coupled to energy storage (not shown in the diagram). Consequently, electron transport from FADH₂ to oxygen drives proton translocation at only two sites of the respiratory chain rather than three.

Figure 7-34 illustrates the redox potential levels at various segments of the respiratory chain. A sharp drop occurs across each of the three major respiratory complexes. The potential difference between any two electron carriers is directly proportional to The energy released when an electron moves from one carrier to the other (Fig. 7-34). Each complex functions as an energy-transducing device, channeling this free energy into the translocation of protons across the membrane, thereby generating an electrochemical proton gradient as electrons traverse the chain. This energy conversion can be directly demonstrated by incorporating any isolated respiratory chain complex individually into liposomes (see Fig. 7-25). In the presence of a suitable electron donor and acceptor, such a complex will mediate electron transport, resulting in proton pumping across the liposome membrane.

For the energy-transducing mechanism underlying oxidative phosphorylation to operate, each enzyme complex of the respiratory chain must be oriented within the inner mitochondrial membrane in a specific manner—such that all protons are translocated in the same direction, i.e., from the matrix outward (Fig. 7-35). This vectorial Organization of Membrane proteins has been demonstrated using impermeant chemical probes that label the complex from only one side of the membrane (Section 6.2.3). Such specific orientation within the bilayer is a general property of membrane proteins and is crucial for their function.

Fig. 7-35. Schematic diagram of the transfer of two electrons from NADH to oxygen through the three major respiratory complexes. Ubiquinone and cytochrome c act as mobile carriers between the complexes.

7.2.9. The mechanisms of proton pumping by respiratory chain components remain incompletely understood

During oxidative phosphorylation, The oxidation of a single molecule of NADH (i.e., the passage of two electrons through all three enzyme complexes) yields a maximum of three ATP molecules. Assuming that the reverse translocation of three protons through ATP synthase drives the synthesis of one ATP molecule (Section 7.2.3), one can conclude that, on average, the transfer of a single electron by each complex is accompanied by the translocation of 1.5 protons (in other words, during The transport of one electron, certain complexes pump one proton, whereas others pump two).

Different components of the respiratory chain likely employ distinct mechanisms to couple electron transport to proton translocation. Allosteric Conformational Changes in protein molecules associated with electron transport can, in principle, drive proton "pumping," much like protons are translocated during the reverse reaction of ATP synthase (Section 7.2.3). Furthermore, as previously mentioned, upon accepting each electron, quinone picks up a proton from the aqueous medium, which it subsequently releases when the electron is passed on (see Fig. 7-30). Because ubiquinone diffuses freely within the lipid bilayer, it can accept electrons near the inner membrane surface and deliver them to the b-c₁ complex near the outer surface, thereby transporting one H⁺ ion across the bilayer per transferred electron. More complex models can also account for the translocation of two protons per electron by the b-c₁ complex by proposing that ubiquinone undergoes a specific cyclic pathway through the b-c₁ complex.

In contrast, the molecules that deliver electrons to the cytochrome oxidase complex do not appear to translocate protons. In this case, Electron transport is presumably coupled to a specific allosteric conformational change in the protein molecules, whereby a segment of the protein complex itself mediates proton translocation.

7.2.10. H⁺-ionophores dissipate the proton gradient and thereby uncouple Electron Transport and ATP synthesis [22]

Since the 1940s, A number of lipophilic Weak acids have been known to act as uncoupling agents—that is, they disrupt the coupling between electron transport and ATP synthesis. When these low-molecular-weight Organic compounds are added to cells, mitochondria cease synthesizing ATP while continuing to consume oxygen. In the presence of an uncoupler, The rate of electron transport remains high, but no proton gradient is generated. There is a simple and elegant explanation for this effect: uncoupling agents act as H⁺ carriers (H⁺ ionophores), opening up an alternative pathway—bypassing ATP synthase—for the flow of H+ across the inner mitochondrial membrane. This mechanism is illustrated in Fig. 7-36 using the widely employed uncoupler 2,4-dinitrophenol as an example.

7.2.11. Under normal conditions, electron flow through the respiratory chain is restrained by Respiratory Control [23]

When an uncoupling agent such as dinitrophenol is added to cells, mitochondrial oxygen consumption increases markedly because the rate of electron transfer goes up. This acceleration is related to a phenomenon known as respiratory control. It is believed that this control relies on the direct inhibitory effect of the electrochemical proton gradient on electron transport. When the electrochemical gradient collapses in the presence of an uncoupler, unhindered electron transport reaches its maximum velocity. Conversely, as the gradient builds up, it retards the respiratory chain, slowing down electron transport. Furthermore, if an unusually high electrochemical gradient is artificially established across the inner membrane in an experiment, normal electron transport ceases entirely, and a reverse electron flow can even be detected in certain segments of the respiratory chain. This suggests that respiratory control reflects a delicate balance between the free-energy change associated with proton translocation (which is coupled to electron transport) and the free-energy change of electron transport itself. In other words, the magnitude of the electrochemical gradient influences both the rate and direction of electron transfer, just as it affects the direction of ATP synthase activity (see Section 7.2.3).

Respiratory control is merely part of an intricate network of interconnected feedback regulatory mechanisms that coordinate the rates of Glycolysis, fatty acid breakdown, The Citric Acid cycle, and electron transport. The rates of all these processes depend on the ATP:ADP ratio, increasing when this ratio drops due to enhanced ATP consumption. For instance, the ATP synthase in the inner mitochondrial membrane operates faster when the concentrations of its substrates—namely ADP and P_i—rise. The higher the rate of this reaction, the more protons flow into the matrix, thereby dissipating the electrochemical gradient more rapidly; in turn, the reduction of the gradient accelerates electron transport.

Similar regulatory mechanisms—including the feedback inhibition of several Key Enzymes by adenosine triphosphate (ATP) (see, for example, Fig. 7-13)—coordinate the rate of NADH production with its utilization by the respiratory chain. Thanks to these numerous adaptations, an exercising Organism can oxidize fats and sugars 5 to 10 times faster than it does at rest.

Fig. 7-36. Proton translocation across the inner mitochondrial membrane mediated by the uncoupling agent 2,4-dinitrophenol (DNP). The charged (protonated) form of DNP can freely diffuse across the lipid bilayer. The translocation of the charged form of DNP across the membrane is believed to be facilitated by an anion carrier protein (not shown in the diagram). As illustrated, driven by the electrochemical proton gradient, dinitrophenol molecules will transport more protons into the matrix than out of it until the proton-motive force is completely abolished.

7.2.12. Natural uncouplers turn brown adipose tissue mitochondria into heat generators [24]

In certain specialized cells—specifically those of brown adipose tissue—mitochondrial Respiration can be naturally uncoupled from ATP synthesis, causing most of the oxidation energy to be dissipated as heat rather than being captured in ATP. The inner membrane of the large mitochondria in these cells contains a specialized transport protein that allows protons to flow down their electrochemical gradient without activating ATP synthase. As a consequence, these cells oxidize fat reserves at a high rate, generating substantial amounts of heat and very little ATP. Thus, brown adipose tissue acts as a biological furnace that can rapidly arouse animals from hibernation and protect the most vulnerable parts of a newborn infant's body from hypothermia.

7.2.13. All bacteria utilize chemiosmotic mechanisms

Bacteria extract energy from a remarkably diverse array of sources. Some bacteria, much like animal cells, synthesize ATP by oxidizing sugars to CO and H₂O via glycolysis followed by the citric acid cycle; The Plasma Membrane of such bacteria features a respiratory chain similar to that of the inner mitochondrial membrane. Other types of bacteria are strict anaerobes that derive energy exclusively through glycolysis (Fermentation) or through oxidative processes in which the terminal electron acceptor is a molecule other than oxygen. Such alternative acceptors may include nitrogen compounds (nitrate or nitrite), sulfur compounds (sulfate or sulfite), or carbon compounds (fumarate or carbonate). Electrons are transferred to these acceptors via a series of carriers located in the plasma membrane that resemble the components of the mitochondrial respiratory chain.

Despite all these variations, the plasma membrane of most bacteria contains an ATP synthase very similar to that found in mitochondria (and Chloroplasts). In anaerobes lacking an electron transport chain, ATP synthase operates in reverse, utilizing ATP generated by glycolysis to establish a proton-motive force across the plasma membrane. In aerobic bacteria, the proton-motive force that drives ATP synthesis is generated by the electron transport chain.

Almost all bacteria, including strict anaerobes, maintain a proton-motive force across their membrane. They harness the energy of the electrochemical proton gradient to rotate their flagella for locomotion (Section 12.5.4) and to pump H⁺ ions out of the cell via an antiport mechanism that functionally replaces the Na⁺,K⁺-ATPase of Eukaryotic cells. In animal cells, active uptake of substances across the plasma membrane relies primarily on the sodium gradient generated by the Na⁺,K⁺-ATPase (Section 6.4.9). By contrast, active nutrient uptake in bacteria occurs via H⁺ symport, where essential metabolites enter the cell alongside one or more protons, facilitated by a specific carrier protein. Many sugars and Most Amino Acids are transported into the bacterial cell in this manner (Fig. 7-37).

Fig. 7-37. The proton-motive force generated across the bacterial plasma membrane drives the uptake of nutrients and the extrusion of sodium. In the presence of oxygen (A), the respiratory chain of aerobic bacteria creates an electrochemical proton gradient that is utilized by ATP synthase to synthesize ATP and to drive the uptake of certain nutrients. Under anaerobic conditions (B), these same bacteria obtain ATP through glycolysis. Hydrolysis of a fraction of this ATP by ATP synthase generates a transmembrane proton-motive force that provides energy for transport processes. (As discussed in the text, some bacteria possess Electron Transport Chains that pump out protons even under anaerobic conditions, using an electron acceptor other than oxygen.)

Among the vast diversity of bacteria, some forms have adapted to extremely harsh environments. For instance, certain bacteria thrive in highly alkaline habitats and must maintain intracellular pH values lower than the extracellular environment to protect alkali-sensitive molecules such as RNA. Under such conditions, an inwardly directed proton concentration gradient (where H⁺ concentration is higher inside than outside) would oppose the establishment of an electrochemical proton gradient. Presumably for this reason, alkali-tolerant marine bacteria such as *Vibrio alginolyticus* rely on Na⁺ rather than H⁺ for all their chemiosmotic mechanisms. Their respiratory chain extrudes Na⁺, nutrient uptake is coupled to the influx of Na⁺, flagellar rotation is powered by the inward Na⁺ current, and presumably a large fraction of ATP is synthesized by a Na⁺-driven ATP synthase. The existence of such bacteria demonstrates that THE PRINCIPLE OF chemiosmosis is even more fundamental than its most common manifestation—the utilization of the electrochemical proton gradient.

Conclusion

The respiratory chain of the inner mitochondrial membrane comprises three major enzyme complexes involved in transferring electrons from NADH to O₂. If any of these complexes is isolated and incorporated into a liposome membrane, its ability to translocate protons simultaneously with electron transport can be readily demonstrated. In the native membrane, the electron transport chain is augmented by mobile carriers—ubiquinone and cytochrome *c*—which shuttle back and forth between enzyme complexes. The pathway of electrons through this chain can be represented by the following sequence: NADH → NADH dehydrogenase complex → ubiquinone → *bc*₁ complex → cytochrome *c* → cytochrome oxidase complex → molecular oxygen (O₂).

The respiratory enzyme complexes couple the energy-releasing steps of electron transport to the outward pumping of protons from the matrix. The resulting electrochemical proton gradient provides the energy for ATP synthesis via another transmembrane protein complex, the ATP synthase, which allows protons to flow back into the matrix. ATP synthase is a reversible coupling device; under normal conditions, it harnesses the energy of the inward proton flow to synthesize ATP via phosphate bond formation, but when the electrochemical proton gradient diminishes, it can also use the energy of ATP hydrolysis to pump protons out of the matrix. Chemiosmotic mechanisms are shared by mitochondria, chloroplasts, and bacteria alike, underscoring their exceptional importance across all living cells.