Chapter 14:  Energy Conversion: Mitochondria and Chloroplasts

The Mitochondrion Contains an Outer Membrane, an Inner Membrane, and Two Internal Compartments

Each mitochondrion is bounded by two highly specialized membranes, which have very different functions. Together they create two separate mitochondrial compartments: the internal matrix and a much narrower intermembrane space. If purified mitochondria are gently disrupted and then fractionated into separate components (Figure 14-7), the biochemical composition of each of the two membranes and of the spaces enclosed by them can be determined. As described in Figure 14-8, each contains a unique collection of proteins.

The outer membrane contains many copies of a transport protein called porin (discussed in Chapter 11), which forms large aqueous channels through the lipid bilayer. This membrane thus resembles a sieve that is permeable to all molecules of 5000 daltons or less, including small proteins. Such molecules can enter the intermembrane space, but most of them cannot pass the impermeable inner membrane. Thus, whereas the intermembrane space is chemically equivalent to the cytosol with respect to the small molecules it contains, the matrix contains a highly selected set of these molecules.

As we explain in detail later, the major working part of the mitochondrion is the matrix and the inner membrane that surrounds it. The inner membrane is highly specialized. Its lipid bilayer contains a high proportion of the “double” phospholipid cardiolipin, which has four fatty acids rather than two and may help to make the membrane especially impermeable to ions. This membrane also contains a variety of transport proteins that make it selectively permeable to those small molecules that are metabolized or required by the many mitochondrial enzymes concentrated in the matrix. The matrix enzymes include those that metabolize pyruvate and fatty acids to produce acetyl CoA and those that oxidize acetyl CoA in the citric acid cycle. The principal end-products of this oxidation are CO₂, which is released from the cell as waste, and NADH, which is the main source of electrons for transport along the respiratory chain—the name given to the electron-transport chain in mitochondria. The enzymes of the respiratory chain are embedded in the inner mitochondrial membrane, and they are essential to the process of oxidative phosphorylation, which generates most of the animal cell's ATP.

The inner membrane is usually highly convoluted, forming a series of infoldings, known as cristae, that project into the matrix. These convolutions greatly increase the area of the inner membrane, so that in a liver cell, for example, it constitutes about one-third of the total cell membrane. The number of cristae is three times greater in the mitochondrion of a cardiac muscle cell than in the mitochondrion of a liver cell, presumably because of the greater demand for ATP in heart cells. There are also substantial differences in the mitochondrial enzymes of different cell types. In this chapter, we largely ignore these differences and focus instead on the enzymes and properties that are common to all mitochondria.

High-Energy Electrons Are Generated via the Citric Acid Cycle

As previously mentioned, without mitochondria present-day eucaryotes would be dependent on the relatively inefficient process of glycolysis (described in Chapter 2) for all of their ATP production, and it seems unlikely that complex multicellular organisms could have been supported in this way. When glucose is converted to pyruvate by glycolysis, less than 10% of the total free energy potentially available from the glucose is released. In the mitochondria, the metabolism of sugars is completed, and the energy released is harnessed so efficiently that about 30 molecules of ATP are produced for each molecule of glucose oxidized. By contrast, only 2 molecules of ATP are produced per glucose molecule by glycolysis alone.

Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate comes from glucose and other sugars, whereas fatty acids come from fats. Both of these fuel molecules are transported across the inner mitochondrial membrane and then converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the mitochondrial matrix. The acetyl groups in acetyl CoA are then oxidized in the matrix via the citric acid cycle, described in Chapter 2. The cycle converts the carbon atoms in acetyl CoA to CO₂, which is released from the cell as a waste product. Most importantly, the cycle generates high-energy electrons, carried by the activated carrier molecules NADH and FADH₂ (Figure 14-9). These high-energy electrons are then transferred to the inner mitochondrial membrane, where they enter the electron-transport chain; the loss of electrons from NADH and FADH₂ also regenerates the NAD⁺ and FAD that is needed for continued oxidative metabolism. The entire sequence of reactions is outlined in Figure 14-10.

A Chemiosmotic Process Converts Oxidation Energy into ATP

Although the citric acid cycle is considered to be part of aerobic metabolism, it does not itself use the oxygen. Only in the final catabolic reactions that take place on the inner mitochondrial membrane is molecular oxygen (O₂) directly consumed. Nearly all the energy available from burning carbohydrates, fats, and other foodstuffs in the earlier stages of their oxidation is initially saved in the form of high-energy electrons removed from substrates by NAD⁺ and FAD. These electrons, carried by NADH and FADH₂, are then combined with O₂ by means of the respiratory chain embedded in the inner mitochondrial membrane. The large amount of energy released is harnessed by the inner membrane to drive the conversion of ADP + P_i to ATP. For this reason, the term oxidative phosphorylation is used to describe this last series of reactions (Figure 14-11).

As previously mentioned, the generation of ATP by oxidative phosphorylation via the respiratory chain depends on a chemiosmotic process. When it was first proposed in 1961, this mechanism explained a long-standing puzzle in cell biology. Nonetheless, the idea was so novel that it was some years before enough supporting evidence accumulated to make it generally accepted.

In the remainder of this section we shall briefly outline the type of reactions that make oxidative phosphorylation possible, saving the details of the respiratory chain for later.

Electrons Are Transferred from NADH to Oxygen Through Three Large Respiratory Enzyme Complexes

Although the mechanism by which energy is harvested by the respiratory chain differs from that in other catabolic reactions, the principle is the same. The energetically favorable reaction H₂ + ½O₂ → H₂O is made to occur in many small steps, so that most of the energy released can be stored instead of being lost to the environment as heat. The hydrogen atoms are first separated into protons and electrons. The electrons pass through a series of electron carriers in the inner mitochondrial membrane. At several steps along the way, protons and electrons are transiently recombined. But only when the electrons reach the end of the electron-transport chain are the protons returned permanently, when they are used to neutralize the negative charges created by the final addition of the electrons to the oxygen molecule (Figure 14-12).

The process of electron transport begins when the hydride ion is removed from NADH (to regenerate NAD⁺) and is converted into a proton and two electrons (H^-→ H⁺ + 2e ^-). The two electrons are passed to the first of the more than 15 different electron carriers in the respiratory chain. The electrons start with very high energy and gradually lose it as they pass along the chain. For the most part, the electrons pass from one metal ion to another, each of these ions being tightly bound to a protein molecule that alters the electron affinity of the metal ion (discussed in detail later). Most of the proteins involved are grouped into three large respiratory enzyme complexes, each containing transmembrane proteins that hold the complex firmly in the inner mitochondrial membrane. Each complex in the chain has a greater affinity for electrons than its predecessor, and electrons pass sequentially from one complex to another until they are finally transferred to oxygen, which has the greatest affinity of all for electrons.

As Electrons Move Along the Respiratory Chain, Energy Is Stored as an Electrochemical Proton Gradient Across the Inner Membrane

Oxidative phosphorylation is made possible by the close association of the electron carriers with protein molecules. The proteins guide the electrons along the respiratory chain so that the electrons move sequentially from one enzyme complex to another—with no short circuits. Most importantly, the transfer of electrons is coupled to oriented H⁺ uptake and release, as well as to allosteric changes in energy-converting protein pumps. The net result is the pumping of H⁺ across the inner membrane—from the matrix to the intermembrane space, driven by the energetically favorable flow of electrons. This movement of H⁺ has two major consequences:

• 1

  It generates a pH gradient across the inner mitochondrial membrane, with the pH higher in the matrix than in the cytosol, where the pH is generally close to 7. (Since small molecules equilibrate freely across the outer membrane of the mitochondrion, the pH in the intermembrane space is the same as in the cytosol.)

• 2

  It generates a voltage gradient (membrane potential) across the inner mitochondrial membrane, with the inside negative and the outside positive (as a result of the net outflow of positive ions).

The pH gradient (ΔpH) drives H⁺ back into the matrix and OH^- out of the matrix, thereby reinforcing the effect of the membrane potential (ΔV), which acts to attract any positive ion into the matrix and to push any negative ion out. Together, the ΔpH and the ΔV are said to constitute an electrochemical proton gradient (Figure 14-13).

The electrochemical proton gradient exerts a proton-motive force, which can be measured in units of millivolts (mV). Since each ΔpH of 1 pH unit has an effect equivalent to a membrane potential of about 60 mV, the total proton-motive force equals ΔV - 60(ΔpH). In a typical cell, the proton-motive force across the inner membrane of a respiring mitochondrion is about 200 mV and is made up of a membrane potential of about 140 mV and a pH gradient of about -1 pH unit.

How the Proton Gradient Drives ATP Synthesis

The electrochemical proton gradient across the inner mitochondrial membrane is used to drive ATP synthesis in the critical process of oxidative phosphorylation (Figure 14-14). This is made possible by the membrane-bound enzyme ATP synthase, mentioned previously. This enzyme creates a hydrophilic pathway across the inner mitochondrial membrane that allows protons to flow down their electrochemical gradient. As these ions thread their way through the ATP synthase, they are used to drive the energetically unfavorable reaction between ADP and P_i that makes ATP (see Figure 2-27). The ATP synthase is of ancient origin; the same enzyme occurs in the mitochondria of animal cells, the chloroplasts of plants and algae, and in the plasma membrane of bacteria and archea.

The structure of ATP synthase is shown in Figure 14-15. Also called the F₀F₁ ATPase, it is a multisubunit protein with a mass of more than 500,000 daltons. A large enzymatic portion, shaped like a lollipop head and composed of a ring of 6 subunits, projects on the matrix side of the inner mitochondrial membrane. This head is held in place by an elongated arm that binds to the head, tying it to a group of transmembrane proteins that produce a “stator” in the membrane. This stator is in contact with a “rotor” that is formed by a ring of 10 to 14 identical transmembrane protein subunits. As protons pass through a narrow channel formed at the stator-rotor contact, their movement causes the rotor ring to spin. This spinning also turns a stalk attached to the rotor (blue in Figure 14-15B), which is thereby made to turn rapidly inside the lollipop head. As a result, the energy of proton flow down a gradient has been converted into the mechanical energy of two sets of proteins rubbing against each other: rotating stalk proteins pushing against a stationary ring of head proteins.

Three of the six subunits in the head contain binding sites for ADP and inorganic phosphate. These are driven to form ATP as mechanical energy is converted into chemical bond energy through the repeated changes in protein conformation that the rotating stalk creates. In this way, the ATP synthase is able to produce more than 100 molecules of ATP per second. Three or four protons need to pass through this marvelous device to make each molecule of ATP.

How the Proton Gradient Drives Coupled Transport Across the Inner Membrane

The synthesis of ATP is not the only process driven by the electrochemical proton gradient. In mitochondria, many charged small molecules, such as pyruvate, ADP, and P_i, are pumped into the matrix from the cytosol, while others, such as ATP, must be moved in the opposite direction. Carrier proteins that bind these molecules can couple their transport to the energetically favorable flow of H⁺ into the mitochondrial matrix. Thus, for example, pyruvate and inorganic phosphate (P_i) are co-transported inward with H⁺ as the H⁺ moves into the matrix.

In contrast, ADP is co-transported with ATP in opposite directions by a single carrier protein. Since an ATP molecule has one more negative charge than ADP, each nucleotide exchange results in a total of one negative charge being moved out of the mitochondrion. This ADP-ATP co-transport is thereby driven by the voltage difference across the membrane (Figure 14-16).

In eucaryotic cells, the proton gradient is thus used to drive both the formation of ATP and the transport of certain metabolites across the inner mitochondrial membrane. In bacteria, the proton gradient across the bacterial plasma membrane is harnessed for both types of functions. And in the plasma membrane of motile bacteria, the gradient also drives the rapid rotation of the bacterial flagellum, which propels the bacterium along (Figure 14-17).

Proton Gradients Produce Most of the Cell's ATP

As stated previously, glycolysis alone produces a net yield of 2 molecules of ATP for every molecule of glucose that is metabolized, and this is the total energy yield for the fermentation processes that occur in the absence of O₂ (discussed in Chapter 2). During oxidative phosphorylation, each pair of electrons donated by the NADH produced in mitochondria is thought to provide energy for the formation of about 2.5 molecules of ATP, after subtracting the energy needed for transporting this ATP to the cytosol. Oxidative phosphorylation also produces 1.5 ATP molecules per electron pair from FADH₂, or from the NADH molecules produced by glycolysis in the cytosol. From the product yields of glycolysis and the citric acid cycle summarized in Table 14-1A, one can calculate that the complete oxidation of one molecule of glucose—starting with glycolysis and ending with oxidative phosphorylation—gives a net yield of about 30 ATPs.

In conclusion, the vast majority of the ATP produced from the oxidation of glucose in an animal cell is produced by chemiosmotic mechanisms in the mitochondrial membrane. Oxidative phosphorylation in the mitochondrion also produces a large amount of ATP from the NADH and the FADH₂ that is derived from the oxidation of fats (Table 14-1B; see also Figure 2-78).

Mitochondria Maintain a High ATP:ADP Ratio in Cells

Because of the carrier protein in the inner mitochondrial membrane that exchanges ATP for ADP, the ADP molecules produced by ATP hydrolysis in the cytosol rapidly enter mitochondria for recharging, while the ATP molecules formed in the mitochondrial matrix by oxidative phosphorylation are rapidly pumped into the cytosol, where they are needed. A typical ATP molecule in the human body shuttles out of a mitochondrion and back into it (as ADP) for recharging more than once per minute, keeping the concentration of ATP in the cell about 10 times higher than that of ADP.

As discussed in Chapter 2, biosynthetic enzymes often drive energetically unfavorable reactions by coupling them to the energetically favorable hydrolysis of ATP (see Figure 2-56). The ATP pool is therefore used to drive cellular processes in much the same way that a battery can be used to drive electric engines. If the activity of the mitochondria is blocked, ATP levels fall and the cell's battery runs down; eventually, energetically unfavorable reactions are no longer driven, and the cell dies. The poison cyanide, which blocks electron transport in the inner mitochondrial membrane, causes death in exactly this way.

It might seem that cellular processes would stop only when the concentration of ATP reaches zero; but, in fact, life is more demanding: it depends on cells maintaining a concentration of ATP that is high compared to the concentrations of ADP and P_i. To explain why, we must consider some elementary principles of thermodynamics.

A Large Negative Value of ΔG for ATP Hydrolysis Makes ATP Useful to the Cell

In Chapter 2, the concept of free energy (G) was discussed. The free energy change for a reaction, ΔG, determines whether this reaction will occur in a cell. We showed on p. 80 that the ΔG for a given reaction can be written as the sum of two parts: the first, called the standard free-energy change, ΔG°, depends on the intrinsic characters of the reacting molecules; the second depends on their concentrations. For the simple reaction A → B,

where [A] and [B] denote the concentrations of A and B, and ln is the natural logarithm. ΔG° is therefore only a reference value that equals the value of ΔG when the molar concentrations of A and B are equal (ln 1 = 0).

In Chapter 2, ATP was described as the major “activated carrier molecule” in cells. The large, favorable free-energy change (large negative ΔG) for its hydrolysis is used, through coupled reactions, to drive other chemical reactions that would otherwise not occur (see pp. 82–85). The ATP hydrolysis reaction produces two products, ADP and inorganic phosphate (P_i); it is therefore of the type A → B + C, where, as described in Figure 14-18,

When ATP is hydrolyzed to ADP and P_i under the conditions that normally exist in a cell, the free-energy change is roughly -11 to -13 kcal/mole. This extremely favorable ΔG depends on having a high concentration of ATP in the cell compared with the concentration of ADP and P_i. When ATP, ADP, and P_i are all present at the same concentration of 1 mole/liter (so-called standard conditions), the ΔG for ATP hydrolysis is the standard free-energy change (ΔG°), which is only -7.3 kcal/mole. At much lower concentrations of ATP relative to ADP and P_i, ΔG becomes zero. At this point, the rate at which ADP and P_i will join to form ATP will be equal to the rate at which ATP hydrolyzes to form ADP and P_i. In other words, when ΔG = 0, the reaction is at equilibrium (see Figure 14-18).

It is ΔG, not ΔG°, that indicates how far a reaction is from equilibrium and determines whether it can be used to drive other reactions. Because the efficient conversion of ADP to ATP in mitochondria maintains such a high concentration of ATP relative to ADP and P_i, the ATP-hydrolysis reaction in cells is kept very far from equilibrium and ΔG is correspondingly very negative. Without this large disequilibrium, ATP hydrolysis could not be used to direct the reactions of the cell; for example, many biosynthetic reactions would run backward rather than forward at low ATP concentrations.

ATP Synthase Can Also Function in Reverse to Hydrolyze ATP and Pump H⁺

In addition to harnessing the flow of H⁺ down an electrochemical proton gradient to make ATP, the ATP synthase can work in reverse: it can use the energy of ATP hydrolysis to pump H⁺ across the inner mitochondrial membrane (Figure 14-19). It thus acts as a reversible coupling device, interconverting electrochemical proton gradient and chemical bond energies. The direction of action at any instant depends on the balance between the steepness of the electrochemical proton gradient and the local ΔG for ATP hydrolysis, as we now explain.

Although the exact number of protons needed to make each ATP molecule is not known with certainty, we shall assume that one molecule of ATP is made by the ATP synthase for every 3 protons driven through it. Whether the ATP synthase works in its ATP-synthesizing or its ATP-hydrolyzing direction at any instant depends, in this case, on the exact balance between the favorable free-energy change for moving the three protons across the membrane into the matrix ΔG_3H+ (which is less than zero) and the unfavorable free-energy change for ATP synthesis in the matrix ΔG_{ATP synthesis} (which is greater than zero). As just discussed, the value of ΔG_{ATP synthesis} depends on the exact concentrations of the three reactants ATP, ADP, and P_i in the mitochondrial matrix (see Figure 14-18). The value of ΔG_3H+ in contrast, is directly proportional to the value of the proton-motive force across the inner mitochondrial membrane. The following example will help explain how the balance between these two free-energy changes affects the ATP synthase.

As explained in the legend to Figure 14-19, 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 (ΔG_3H+ = -13.8 kcal/mole). Thus, if the proton-motive force remains constant at 200 mV, the ATP synthase synthesizes ATP until a ratio of ATP to ADP and P_i is reached where ΔG_{ATP synthesis} is just equal to +13.8 kcal/mole (here ΔG_{ATP synthesis} + ΔG_3H+ = 0). At this point there is no further net ATP synthesis or hydrolysis by the ATP synthase.

Suppose 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 ΔG_{ATP synthesis} will decrease (see Figure 14-18), 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, ΔG_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 ΔG_{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 same type of reversibility is shared by other membrane transport proteins that couple the transmembrane movement of an ion to ATP synthesis or hydrolysis. Both the Na⁺-K⁺ pump and the Ca²⁺ pump described in Chapter 11, for example, normally hydrolyze ATP and use the energy released to move their 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, the ATP synthase is by no means unique in its ability to convert the electrochemical energy stored in a transmembrane ion gradient directly into phosphate-bond energy in ATP.

Summary

The mitochondrion performs most cellular oxidations and produces the bulk of the animal cell's ATP. The mitochondrial matrix contains a large variety of enzymes, including those that convert pyruvate and fatty acids to acetyl CoA and those that oxidize this acetyl CoA to CO₂through the citric acid cycle. Large amounts of NADH (and FADH₂) are produced by these oxidation reactions.

The energy available from combining molecular oxygen with the reactive electrons carried by NADH and FADH₂ is harnessed by an electron-transport chain in the inner mitochondrial membrane called the respiratory chain. The respiratory chain pumps H⁺ out of the matrix to create a transmembrane electrochemical proton (H⁺) gradient, which includes contributions from both a membrane potential and a pH difference. The large amount of free energy released when H⁺ flows back into the matrix (across the inner membrane) provides the basis for ATP production in the matrix by a remarkable protein machine—the ATP synthase. The transmembrane electrochemical gradient is also used to drive the active transport of selected metabolites across the mitochondrial inner membrane, including an efficient ATP-ADP exchange between the mitochondrion and the cytosol that keeps the cell's ATP pool highly charged. The resulting high ratio of ATP to its hydrolysis products makes the free-energy change for ATP hydrolysis extremely favorable, allowing this hydrolysis reaction to drive a large number of the cell's energy-requiring processes.

Protons Are Unusually Easy to Move

Although protons resemble other positive ions such as Na⁺ and K⁺ in their movement across membranes, in some respects they are unique. Hydrogen atoms are by far the most abundant type of atom in living organisms; they are plentiful not only in all carbon-containing biological molecules, but also in the water molecules that surround them. The protons in water are highly mobile, flickering through the hydrogen-bonded network of water molecules by rapidly dissociating from one water molecule to associate with its neighbor, as illustrated in Figure 14-20A. Protons are thought to move across a protein pump embedded in a lipid bilayer in a similar way: they transfer from one amino acid side chain to another, following a special channel through the protein.

Protons are also special with respect to electron transport. Whenever a molecule is reduced by acquiring an electron, the electron (e ^-) brings with it a negative charge. In many cases, this charge is rapidly neutralized by the addition of a proton (H⁺) from water, so that the net effect of the reduction is to transfer an entire hydrogen atom, H⁺ + e ^- (Figure 14-20B). Similarly, when a molecule is oxidized, a hydrogen atom removed from it can be readily dissociated into its constituent electron and proton—allowing the electron to be transferred separately to a molecule that accepts electrons, while the proton is passed to the water. Therefore, in a membrane in which electrons are being passed along an electron-transport chain, pumping protons from one side of the membrane to another can be relatively simple. The electron carrier merely needs to be arranged in the membrane in a way that causes it to pick up a proton from one side of the membrane when it accepts an electron, and to release the proton on the other side of the membrane as the electron is passed to the next carrier molecule in the chain (Figure 14-21).

The Redox Potential Is a Measure of Electron Affinities

In biochemical reactions, any electrons removed from one molecule are always passed to another, so that whenever one molecule is oxidized, another is reduced. Like any other chemical reaction, the tendency of such oxidation-reduction reactions, or redox reactions, to proceed spontaneously depends on the free-energy change (ΔG) for the electron transfer, which in turn depends on the relative affinities of the two molecules for electrons.

Because electron transfers provide most of the energy for living things, it is worth spending the time to understand them. Many readers are already familiar with acids and bases, which donate and accept protons (see Panel 2-2, pp. 112–113). Acids and bases exist in conjugate acid-base pairs, in which the acid is readily converted into the base by the loss of a proton. For example, acetic acid (CH₃COOH) is converted into its conjugate base (CH₃COO^-) in the reaction:

In exactly the same way, pairs of compounds such as NADH and NAD⁺ are called redox pairs, since NADH is converted to NAD⁺ by the loss of electrons in the reaction:

NADH is a strong electron donor: because its electrons are held in a high-energy linkage, the free-energy change for passing its electrons to many other molecules is favorable (see Figure 14-9). It is difficult to form a high-energy linkage. Therefore its redox partner, NAD⁺, is of necessity a weak electron acceptor.

The tendency to transfer electrons from any redox pair can be measured experimentally. All that is required is the formation of an electrical circuit linking a 1:1 (equimolar) mixture of the redox pair to a second redox pair that has been arbitrarily selected as a reference standard, so the voltage difference can be measured between them (Panel 14-1, p. 784). This voltage difference is defined as the redox potential; as defined, electrons move spontaneously from a redox pair like NADH/NAD⁺ with a low redox potential (a low affinity for electrons) to a redox pair like O₂/H₂O with a high redox potential (a high affinity for electrons). Thus, NADH is a good molecule for donating electrons to the respiratory chain, while O₂ is well suited to act as the “sink” for electrons at the end of the pathway. As explained in Panel 14-1, the difference in redox potential, ΔE₀′, is a direct measure of the standard free-energy change (ΔG°) for the transfer of an electron from one molecule to another.

Electron Transfers Release Large Amounts of Energy

As just discussed, 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. Conversely, those pairs that have the most positive redox potentials have the strongest affinity for electrons and therefore contain carriers with the strongest tendency to accept electrons. A 1:1 mixture of NADH and NAD⁺ has a redox potential of -320 mV, indicating that NADH has a strong tendency to donate electrons; a 1:1 mixture of H₂O and ½O₂ has a redox potential of +820 mV, indicating that O₂ has a strong tendency to accept electrons. The difference in redox potential is 1.14 volts (1140 mV), which means that the transfer of each electron from NADH to O₂ under these standard conditions is enormously favorable, where ΔG° = -26.2 kcal/mole (-52.4 kcal/mole for the two electrons transferred per NADH molecule; see Panel 14-1). If we compare this free-energy change with that for the formation of the phosphoanhydride bonds in ATP (ΔG° = -7.3 kcal/mole, see Figure 2-75), we see that more than enough energy is released by the oxidization of one NADH molecule to synthesize several molecules of ATP from ADP and P_i.

Living systems could certainly have evolved enzymes that would allow NADH to donate electrons directly to O₂ to make water in the reaction:

But because of the huge free-energy drop, this reaction would proceed with almost explosive force and nearly all of the energy would be released as heat. Cells do perform this reaction, but they make it proceed much more gradually by passing the high-energy electrons from NADH to O₂ via the many electron carriers in the electron-transport chain. Since each successive carrier in the chain holds its electrons more tightly, the highly energetically favorable reaction 2H⁺ + 2e ^- + ½O₂ → H₂O is made to occur in many small steps. This enables nearly half of the released energy to be stored, instead of being lost to the environment as heat.

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

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 are 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 oxidation state (Fe³⁺) to the ferrous oxidation state (Fe²⁺) 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 14-22). A similar porphyrin ring is responsible for the red color of blood and for the green color of leaves, being bound to iron in hemoglobin and to magnesium in chlorophyll, respectively.

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 14-23). 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 completely characterized. Like the cytochromes, these centers carry one electron at a time.

The simplest of the electron carriers in the respiratory chain—and the only one that is not part of a protein—is a small hydrophobic molecule that is freely mobile in the lipid bilayer known as ubiquinone, or coenzyme Q. A quinone (Q) can pick up or donate either one or two electrons; upon reduction, it picks up a proton from the medium along with each electron it carries (Figure 14-24).

In addition to six different hemes linked to cytochromes, more than seven 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. This pathway involves more than 60 different proteins in all.

As one would expect, the electron carriers have higher and higher affinities for electrons (greater redox potentials) as one moves along the respiratory chain. The redox potentials have been fine-tuned during evolution by the binding of each electron carrier in a particular protein context, which can alter its normal affinity for electrons. However, because iron-sulfur centers have a relatively low affinity for electrons, they predominate in the early part of the respiratory chain; in contrast, the cytochromes predominate further down the chain, where a higher affinity for electrons is required.

The order of the individual electron carriers in the chain was determined by sophisticated spectroscopic measurements (Figure 14-25), 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.

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

Membrane proteins are difficult to purify as intact complexes because they are insoluble in 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, can solubilize selected components of the inner mitochondrial 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 14-26). As we shall see in this section, each of these complexes acts as an electron-transport-driven H⁺ pump; however, they were initially characterized in terms of the electron carriers that they interact with and contain:

• 1

  The NADH dehydrogenase complex (generally known as complex I) is the largest of the respiratory enzyme complexes, containing more than 40 polypeptide chains. It accepts electrons from NADH and passes them through a flavin and at least seven iron-sulfur centers to ubiquinone. Ubiquinone then transfers its electrons to a second respiratory enzyme complex, the cytochrome b-c₁ complex.

• 2

  The cytochrome b-c₁ complex contains at least 11 different polypeptide chains and functions as a dimer. 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.

• 3

  The cytochrome oxidase complex also functions as a dimer; each monomer contains 13 different polypeptide chains, including two cytochromes and two copper atoms. The complex accepts one electron at a time from cytochrome c and passes them four at a time 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. The most obvious of these is cytochrome oxidase.

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

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. The ability of 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; this fact 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 rapidly takes 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, 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 14-27).

The cytochrome oxidase reaction is estimated to account for 90% of the total oxygen uptake in most cells. This protein complex is therefore crucial for all aerobic life. Cyanide and azide are extremely toxic because they bind tightly to the cell's cytochrome oxidase complexes to stop electron transport, thereby greatly reducing ATP production.

Although the cytochrome oxidase in mammals contains 13 different protein subunits, most of these seem 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. The complete structure of this large enzyme complex has recently been determined by x-ray crystallography, as illustrated in Figure 14-28. The atomic resolution structures, combined with mechanistic studies of the effect of precisely tailored mutations introduced into the enzyme by genetic engineering of the yeast and bacterial proteins, are revealing the detailed mechanisms of this finely tuned protein machine.

Electron Transfers Are Mediated by Random Collisions in the Inner Mitochondrial Membrane

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 more slowly diffusing enzyme complexes can account for the observed rates of electron transfer (each complex donates and receives an electron about once every 5–20 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 seem to exist as independent entities in the plane of the inner membrane, being present in different ratios in different mitochondria.

The ordered transfer of electrons along the respiratory chain is due entirely to the specificity of the functional interactions between the components of the chain: each electron carrier is able to interact only with the carrier adjacent to it in the sequence shown in Figure 14-26, with no short circuits.

Electrons move between the molecules that carry them in biological systems not only by moving along covalent bonds within a molecule, but also by jumping across a gap as large as 2 nm. The jumps occur by electron “tunneling,” a quantum-mechanical property that is critical for the processes we are discussing. Insulation is needed to prevent short circuits that would otherwise occur when an electron carrier with a low redox potential collides with a carrier with a high redox potential. This insulation seems to be provided by carrying an electron deep enough inside a protein to prevent its tunneling interactions with an inappropriate partner.

How the changes in redox potential from one electron carrier to the next are harnessed to pump protons out of the mitochondrial matrix is the topic we discuss next.

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

We have previously discussed how the redox potential reflects electron affinities (see p. 783). An outline of the redox potentials measured along the respiratory chain is shown in Figure 14-29. These potentials drop in three large steps, one across each major respiratory complex. The change in redox potential between any two electron carriers is directly proportional to the free energy released when an electron transfers between them. Each enzyme complex acts as an energy-conversion device by harnessing some of this free-energy change to pump H⁺ across the inner membrane, thereby creating an electrochemical proton gradient as electrons pass through that complex. This conversion can be demonstrated by purifying each respiratory enzyme complex and incorporating it separately into liposomes: when an appropriate electron donor and acceptor are added so that electrons can pass through the complex, H⁺ is translocated across the liposome membrane.

The Mechanism of H⁺ Pumping Will Soon Be Understood in Atomic Detail

Some respiratory enzyme complexes pump one H⁺ per electron across the inner mitochondrial membrane, whereas others pump two. The detailed mechanism by which electron transport is coupled to H⁺ pumping is different for the three different enzyme complexes. In the cytochrome b-c₁ complex, the quinones clearly have a role. 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 14-24). 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 cytochrome b-c₁ complex near the outside surface, thereby transferring one H⁺ across the bilayer for every electron transported. Two protons are pumped per electron in the cytochrome b-c₁ complex, however, and there is good 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. Exactly how this occurs can now be worked out at the atomic level, because the complete structure of the cytochrome b-c₁ complex has been determined by x-ray crystallography (Figure 14-30).

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 sequential allosteric changes in protein conformation that cause a portion of the protein to pump H⁺ across the mitochondrial inner membrane. A general mechanism for this type of H⁺ pumping is presented in Figure 14-31.

H⁺ Ionophores Uncouple Electron Transport from ATP Synthesis

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 they provide a pathway for the flow of H⁺ across the inner mitochondrial membrane that bypasses the ATP synthase. 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

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 14-19).

Respiratory control is just one part of an elaborate interlocking system of feedback controls that coordinate 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 an 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, 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 5–10 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

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, by-passing 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 serve as “heating pads,” helping to revive hibernating animals and to protect sensitive areas of newborn human babies from the cold.

Bacteria Also Exploit Chemiosmotic Mechanisms to Harness Energy

Bacteria use enormously diverse energy sources. Some, like animal cells, are aerobic; they synthesize ATP from sugars they oxidize to CO₂ and H₂O by glycolysis, the citric acid cycle, and a respiratory chain in their plasma membrane that is similar to the one in the inner mitochondrial membrane. Others are strict anaerobes, deriving their energy either from glycolysis alone (by fermentation) or 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 the one in mitochondria. In bacteria that use an electron-transport chain to harvest energy, the electron-transport pumps H⁺ out of the cell and thereby establishes a proton-motive force across the plasma membrane that drives the ATP synthase to make ATP. In other bacteria, the ATP synthase works in reverse, using the ATP produced by glycolysis to pump H⁺ and establish a proton gradient across the plasma membrane. The ATP used for this process is generated by fermentation processes (discussed in Chapter 2).

Thus, most bacteria, including the strict anaerobes, maintain a proton gradient across their plasma membrane. It can be harnessed to drive a flagellar motor, and it is used to pump Na⁺ out of the bacterium via a Na⁺-H⁺ antiporter that takes the place of the Na⁺-K⁺ pump of eucaryotic cells. This gradient is also used for the active inward transport of nutrients, such as most amino acids and many sugars: each nutrient is dragged into the cell along with one or more H⁺ through a specific symporter (Figure 14-32). In animal cells, by contrast, most inward transport across the plasma membrane is driven by the Na⁺ gradient that is established by the Na⁺-K⁺ pump.

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, 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 respiratory 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 intact 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 → cytochrome 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 sufficiently reduced. Its universal presence in mitochondria, chloroplasts, and procaryotes testifies to the central importance of chemiosmotic mechanisms in cells.

The Chloroplast Is One Member of the Plastid Family of Organelles

Chloroplasts are the most prominent members of the plastid family of organelles. Plastids are present in all living plant cells, each cell type having its own characteristic complement. All plastids share certain features. Most notably, all plastids in a particular plant species contain multiple copies of the same relatively small genome. In addition, each is enclosed by an envelope composed of two concentric membranes.

As discussed in Chapter 12 (see Figure 12-3), all plastids develop from proplastids, small organelles in the immature cells of plant meristems (Figure 14-33A). Proplastids develop according to the requirements of each differentiated cell, and the type that is present is determined in large part by the nuclear genome. If a leaf is grown in darkness, its proplastids enlarge and develop into etioplasts, which have a semicrystalline array of internal membranes containing a yellow chlorophyll precursor instead of chlorophyll. When exposed to light, the etioplasts rapidly develop into chloroplasts by converting this precursor to chlorophyll and by synthesizing new membrane pigments, photosynthetic enzymes, and components of the electron-transport chain.

Leucoplasts are plastids present in many epidermal and internal tissues that do not become green and photosynthetic. They are little more than enlarged proplastids. A common form of leucoplast is the amyloplast (Figure 14-33B), which accumulates the polysaccharide starch in storage tissues—a source of sugar for future use. In some plants, such as potatoes, the amyloplasts can grow to be as large as an average animal cell.

It is important to realize that plastids are not just sites for photosynthesis and the deposition of storage materials. Plants have also used their plastids to compartmentalize their intermediary metabolism. Purine and pyrimidine synthesis, most amino acid synthesis, and all of the fatty acid synthesis of plants takes place in the plastids, whereas in animal cells these compounds are produced in the cytosol.

Chloroplasts Resemble Mitochondria But Have an Extra Compartment

Chloroplasts carry out their energy interconversions by chemiosmotic mechanisms in much the same way that mitochondria do. Although much larger (Figure 14-34A), they are organized on the same principles. They have a highly permeable outer membrane; a much less permeable inner membrane, in which membrane transport proteins are embedded; and a narrow intermembrane space in between. Together, these membranes form the chloroplast envelope (Figure 14-34B,C). The inner membrane surrounds a large space called the stroma, which is analogous to the mitochondrial matrix and contains many metabolic enzymes. Like the mitochondrion, the chloroplast has its own genome and genetic system. The stroma therefore also contains a special set of ribosomes, RNAs, and the chloroplast DNA.

There is, however, an important difference between the organization of mitochondria and that of chloroplasts. The inner membrane of the chloroplast is not folded into cristae and does not contain electron-transport chains. Instead, the electron-transport chains, photosynthetic light-capturing systems, and ATP synthase are all contained in the thylakoid membrane, a third distinct membrane that forms a set of flattened disclike sacs, the thylakoids (Figure 14-35). The lumen of each thylakoid is thought to be connected with the lumen of other thylakoids, thereby defining a third internal compartment called the thylakoid space, which is separated by the thylakoid membrane from the stroma that surrounds it.

The structural similarities and differences between mitochondria and chloroplasts are illustrated in Figure 14-36. The head of the chloroplast ATP synthase, where ATP is made, protrudes from the thylakoid membrane into the stroma, whereas it protrudes into the matrix from the inner mitochondrial membrane.

Chloroplasts Capture Energy from Sunlight and Use It to Fix Carbon

The many reactions that occur during photosynthesis in plants can be grouped into two broad categories:

• 1

  In the photosynthetic electron-transfer reactions (also called the “light reactions”), energy derived from sunlight energizes an electron in the green organic pigment chlorophyll, enabling the electron to move along an electron-transport chain in the thylakoid membrane in much the same way that an electron moves along the respiratory chain in mitochondria. The chlorophyll obtains its electrons from water (H₂O), producing O₂ as a by-product. During the electron-transport process, H⁺ is pumped across the thylakoid membrane, and the resulting electrochemical proton gradient drives the synthesis of ATP in the stroma. As the final step in this series of reactions, high-energy electrons are loaded (together with H⁺) onto NADP⁺, converting it to NADPH. All of these reactions are confined to the chloroplast.

• 2

  In the carbon-fixation reactions (also called the “dark reactions”), the ATP and the NADPH produced by the photosynthetic electron-transfer reactions serve as the source of energy and reducing power, respectively, to drive the conversion of CO₂ to carbohydrate. The carbon-fixation reactions, which begin in the chloroplast stroma and continue in the cytosol, produce sucrose and many other organic molecules in the leaves of the plant. The sucrose is exported to other tissues as a source of both organic molecules and energy for growth.

Thus, the formation of ATP, NADPH, and O₂ (which requires light energy directly) and the conversion of CO₂ to carbohydrate (which requires light energy only indirectly) are separate processes (Figure 14-37), although elaborate feedback mechanisms interconnect the two. Several of the chloroplast enzymes required for carbon fixation, for example, are inactivated in the dark and reactivated by light-stimulated electron-transport processes.

Carbon Fixation Is Catalyzed by Ribulose Bisphosphate Carboxylase

We have seen earlier in this chapter how cells produce ATP by using the large amount of free energy released when carbohydrates are oxidized to CO₂ and H₂O. Clearly, therefore, the reverse reaction, in which CO₂ and H₂O combine to make carbohydrate, must be a very unfavorable one that can only occur if it is coupled to other, very favorable reactions that drive it.

The central reaction of carbon fixation, in which an atom of inorganic carbon is converted to organic carbon, is illustrated in Figure 14-38: CO₂ from the atmosphere combines with the five-carbon compound ribulose 1,5-bisphosphate plus water to yield two molecules of the three-carbon compound 3-phosphoglycerate. This “carbon-fixing” reaction, which was discovered in 1948, is catalyzed in the chloroplast stroma by a large enzyme called ribulose bisphosphate carboxylase. Since each molecule of the complex works sluggishly (processing only about 3 molecules of substrate per second compared to 1000 molecules per second for a typical enzyme), many enzyme molecules are needed. Ribulose bisphosphate carboxylase often constitutes more than 50% of the total chloroplast protein, and it is thought to be the most abundant protein on Earth.

Three Molecules of ATP and Two Molecules of NADPH Are Consumed for Each CO₂ Molecule That Is Fixed

The actual reaction in which CO₂ is fixed is energetically favorable because of the reactivity of the energy-rich compound ribulose 1,5-bisphosphate, to which each molecule of CO₂ is added (see Figure 14-38). The elaborate metabolic pathway that produces ribulose 1,5-bisphosphate requires both NADPH and ATP; it was worked out in one of the first successful applications of radioisotopes as tracers in biochemistry. This carbon-fixation cycle (also called the Calvin cycle) is outlined in Figure 14-39. It starts when 3 molecules of CO₂ are fixed by ribulose bisphosphate carboxylase to produce 6 molecules of 3-phosphoglycerate (containing 6 × 3 = 18 carbon atoms in all: 3 from the CO₂ and 15 from ribulose 1,5-bisphosphate). The 18 carbon atoms then undergo a cycle of reactions that regenerates the 3 molecules of ribulose 1,5-bisphosphate used in the initial carbon-fixation step (containing 3 × 5 = 15 carbon atoms). This leaves 1 molecule of glyceraldehyde 3-phosphate (3 carbon atoms) as the net gain.

A total of 3 molecules of ATP and 2 molecules of NADPH are consumed for each CO₂ molecule converted into carbohydrate. The net equation is:

Thus, both phosphate-bond energy (as ATP) and reducing power (as NADPH) are required for the formation of organic molecules from CO₂ and H₂O. We return to this important point later.

The glyceraldehyde 3-phosphate produced in chloroplasts by the carbon-fixation cycle is a three-carbon sugar that also serves as a central intermediate in glycolysis. Much of it is exported to the cytosol, where it can be converted into fructose 6-phosphate and glucose 1-phosphate by the reversal of several reactions in glycolysis (see Panel 2-8, pp. 124–125). The glucose 1-phosphate is then converted to the sugar nucleotide UDP-glucose, and this combines with the fructose 6-phosphate to form sucrose phosphate, the immediate precursor of the disaccharide sucrose. Sucrose is the major form in which sugar is transported between plant cells: just as glucose is transported in the blood of animals, sucrose is exported from the leaves via vascular bundles, providing the carbohydrate required by the rest of the plant.

Most of the glyceraldehyde 3-phosphate that remains in the chloroplast is converted to starch in the stroma. Like glycogen in animal cells, starch is a large polymer of glucose that serves as a carbohydrate reserve (see Figure 14-33B). The production of starch is regulated so that it is produced and stored as large grains in the chloroplast stroma during periods of excess photosynthetic capacity. This occurs through reactions in the stroma that are the reverse of those in glycolysis: they convert glyceraldehyde 3-phosphate to glucose 1-phosphate, which is then used to produce the sugar nucleotide ADP-glucose, the immediate precursor of starch. At night the starch is broken down to help support the metabolic needs of the plant. Starch provides an important part of the diet of all animals that eat plants.

Carbon Fixation in Some Plants Is Compartmentalized to Facilitate Growth at Low CO₂ Concentrations

Although ribulose bisphosphate carboxylase preferentially adds CO₂ to ribulose 1,5-bisphosphate, it can use O₂ as a substrate in place of CO₂, and if the concentration of CO₂ is low, it will add O₂ to ribulose 1,5-bisphosphate instead (see Figure 14-38). This is the first step in a pathway called photorespiration, whose ultimate effect is to use up O₂ and liberate CO₂ without the production of useful energy stores. In many plants, about one-third of the CO₂ fixed is lost again as CO₂ because of photorespiration.

Photorespiration can be a serious liability for plants in hot, dry conditions, which cause them to close their stomata (the gas exchange pores in their leaves) to avoid excessive water loss. This in turn causes the CO₂ levels in the leaf to fall precipitously, thereby favoring photorespiration. A special adaptation, however, occurs in the leaves of many plants, such as corn and sugar cane that live in hot, dry environments. In these plants, the carbon-fixation cycle occurs only in the chloroplasts of specialized bundle-sheath cells, which contain all of the plant's ribulose bisphosphate carboxylase. These cells are protected from the air and are surrounded by a specialized layer of mesophyll cells that use the energy harvested by their chloroplasts to “pump” CO₂ into the bundle-sheath cells. This supplies the ribulose bisphosphate carboxylase with a high concentration of CO₂, thereby greatly reducing photorespiration.

The CO₂ pump is produced by a reaction cycle that begins in the cytosol of the mesophyll cells. A CO₂-fixation step is catalyzed by an enzyme that binds carbon dioxide (as bicarbonate) and combines it with an activated three-carbon molecule to produce a four-carbon molecule. The four-carbon molecule diffuses into the bundle-sheath cells, where it is broken down to release the CO₂ and generate a molecule with three carbons. The pumping cycle is completed when this three-carbon molecule is returned to the mesophyll cells and converted back to its original activated form. Because the CO₂ is initially captured by converting it into a compound containing four carbons, the CO₂-pumping plants are called C₄ plants. All other plants are called C₃ plants because they capture CO₂ into the three-carbon compound 3-phosphoglycerate (Figure 14-40).

As for any vectorial transport process, pumping CO₂ into the bundle-sheath cells in C₄ plants costs energy. In hot, dry environments, however, this cost can be much less than the energy lost by photorespiration in C₃ plants, so C₄ plants have a potential advantage. Moreover, because C₄ plants can perform photosynthesis at a lower concentration of CO₂ inside the leaf, they need to open their stomata less often and therefore can fix about twice as much net carbon as C₃ plants per unit of water lost. Although the vast majority of plant species are C₃ plants, C₄ plants such as corn and sugar cane are much more effective at converting sunlight energy into biomass than C₃ plants such as cereal grains. They are therefore of special importance in world agriculture.

Photosynthesis Depends on the Photochemistry of Chlorophyll Molecules

Having discussed the carbon-fixation reactions, we now return to the question of how the photosynthetic electron-transfer reactions in the chloroplast generate the ATP and the NADPH needed to drive the production of carbohydrates from CO₂ and H₂O. The required energy is derived from sunlight absorbed by chlorophyll molecules (Figure 14-41). The process of energy conversion begins when a chlorophyll molecule is excited by a quantum of light (a photon) and an electron is moved from one molecular orbital to another of higher energy. As illustrated in Figure 14-42, such an excited molecule is unstable and tends to return to its original, unexcited state in one of three ways:

• 1

  By converting the extra energy into heat (molecular motions) or to some combination of heat and light of a longer wavelength (fluorescence), which is what happens when light energy is absorbed by an isolated chlorophyll molecule in solution.

• 2

  By transferring the energy—but not the electron—directly to a neighboring chlorophyll molecule by a process called resonance energy transfer.

• 3

  By transferring the high-energy electron to another nearby molecule, an electron acceptor, and then returning to its original state by taking up a low-energy electron from some other molecule, an electron donor.

The last two mechanisms are exploited in the process of photosynthesis.

A Photosystem Consists of a Reaction Center Plus an Antenna Complex

Multiprotein complexes called photosystems catalyze the conversion of the light energy captured in excited chlorophyll molecules to useful forms. A photosystem consists of two closely linked components: an antenna complex, consisting of a large set of pigment molecules that capture light energy and feed it to the reaction center; and a photochemical reaction center, consisting of a complex of proteins and chlorophyll molecules that enable light energy to be converted into chemical energy (Figure 14-43).

The antenna complex is important for capturing light. In chloroplasts it consists of a number of distinct membrane protein complexes (known as light-harvesting complexes); together, these proteins bind several hundred chlorophyll molecules per reaction center, orienting them precisely in the thylakoid membrane. Depending on the plant, different amounts of accessory pigments called carotenoids, which protect the chlorophylls from oxidation and can help collect light of other wavelengths, are also located in each complex. When a chlorophyll molecule in the antenna complex is excited, the energy is rapidly transferred from one molecule to another by resonance energy transfer until it reaches a special pair of chlorophyll molecules in the photochemical reaction center. Each antenna complex thereby acts as a funnel, collecting light energy and directing it to a specific site where it can be used effectively (see Figure 14-43).

The photochemical reaction center is a transmembrane protein-pigment complex that lies at the heart of photosynthesis. It is thought to have evolved more than 3 billion years ago in primitive photosynthetic bacteria. The special pair of chlorophyll molecules in the reaction center acts as an irreversible trap for excitation quanta because its excited electron is immediately passed to a chain of electron acceptors that are precisely positioned as neighbors in the same protein complex (Figure 14-44). By moving the high-energy electron rapidly away from the chlorophylls, the photochemical reaction center transfers it to an environment where it is much more stable. The electron is thereby suitably positioned for subsequent reactions, which require more time to complete.

In a Reaction Center, Light Energy Captured by Chlorophyll Creates a Strong Electron Donor from a Weak One

The electron transfers involved in the photochemical reactions just outlined have been analyzed extensively by rapid spectroscopic methods. An enormous amount of detailed information is available for the photosystem of purple bacteria, which is somewhat simpler than the evolutionarily related photosystems in chloroplasts. The reaction center in this photosystem is a large protein-pigment complex that can be solubilized with detergent and purified in active form. In 1985, its complete three-dimensional structure was determined by x-ray crystallography (see Figure 10-38). This structure, combined with kinetic data, provides the best picture we have of the initial electron-transfer reactions that underlie photosynthesis.

The sequence of electron transfers that take place in the reaction center of purple bacteria is shown in Figure 14-45. As outlined previously for the general case (see Figure 14-43), light causes a net electron transfer from a weak electron donor (a molecule with a strong affinity for electrons) to a molecule that is a strong electron donor in its reduced form. The excitation energy in chlorophyll that would normally be released as fluorescence or heat is thereby used instead to create a strong electron donor (a molecule carrying a high-energy electron) where none had been before. In the purple bacterium, the weak electron donor used to fill the electron-deficient hole created by a light-induced charge separation is a cytochrome (see orange box in Figure 14-45); the strong electron donor produced is a quinone. In the chloroplasts of higher plants, a quinone is similarly produced. However, as we discuss next, water serves as the initial weak electron donor, which is why oxygen gas is released by photosynthesis in plants.

Noncyclic Photophosphorylation Produces Both NADPH and ATP

Photosynthesis in plants and cyanobacteria produces both ATP and NADPH directly by a two-step process called noncyclic photophosphorylation. Because two photosystems—called photosystems I and II—are used in series to energize an electron, the electron can be transferred all the way from water to NADPH. As the high-energy electrons pass through the coupled photosystems to generate NADPH, some of their energy is siphoned off for ATP synthesis.

The first of the two photosystems—paradoxically called photosystem II for historical reasons—has the unique ability to withdraw electrons from water. The oxygens of two water molecules bind to a cluster of manganese atoms in a poorly understood water-splitting enzyme. This enzyme enables electrons to be removed one at a time from the water, as required to fill the electron-deficient holes created by light in chlorophyll molecules in the reaction center. As soon as four electrons have been removed from the two water molecules (requiring four quanta of light), O₂ is released. Photosystem II thus catalyzes the reaction 2H₂O + 4 photons → 4H⁺ + 4e ^- + O₂. As we discussed for the electron-transport chain in mitochondria, which uses O₂ and produces water, the mechanism ensures that no partly oxidized water molecules are released as dangerous, highly reactive oxygen radicals. Essentially all the oxygen in the Earth's atmosphere has been produced in this way.

The core of the reaction center in photosystem II is homologous to the bacterial reaction center just described, and it likewise produces strong electron donors in the form of reduced quinone molecules dissolved in the lipid bilayer of the membrane. The quinones pass their electrons to a H⁺ pump called the cytochrome b₆-f complex, which resembles the cytochrome b-c₁ complex in the respiratory chain of mitochondria. The cytochrome b₆-f complex pumps H⁺ into the thylakoid space across the thylakoid membrane (or out of the cytosol across the plasma membrane in cyanobacteria), and the resulting electrochemical gradient drives the synthesis of ATP by an ATP synthase (Figure 14-46).

The final electron acceptor in this electron-transport chain is the second photosystem, photosystem I, which accepts an electron into the electron-deficient hole created by light in the chlorophyll molecule in its reaction center. Each electron that enters photosystem I is finally boosted to a very high-energy level that allows it to be passed to the iron-sulfur center in ferredoxin and then to NADP⁺ to generate NADPH (Figure 14-47).

The scheme for photosynthesis just discussed is known as the Z scheme. By means of its two electron-energizing steps, one catalyzed by each photosystem, an electron is passed from water, which normally holds on to its electrons very tightly (redox potential = +820 mV), to NADPH, which normally holds on to its electrons loosely (redox potential = -320 mV). There is not enough energy in a single quantum of visible light to energize an electron all the way from the bottom of photosystem II to the top of photosystem I, which is presumably the energy change required to pass an electron efficiently from water to NADP⁺. The use of two separate photosystems in series means that the energy from two quanta of light is available for this purpose. In addition, there is enough energy left over to enable the electron-transport chain that links the two photosystems to pump H⁺ across the thylakoid membrane (or the plasma membrane of cyanobacteria), so that the ATP synthase can harness some of the light-derived energy for ATP production.

Chloroplasts Can Make ATP by Cyclic Photophosphorylation Without Making NADPH

In the noncyclic photophosphorylation scheme just discussed, high-energy electrons leaving photosystem II are harnessed to generate ATP and are passed on to photosystem I to drive the production of NADPH. This produces slightly more than 1 molecule of ATP for every pair of electrons that passes from H₂O to NADP⁺ to generate a molecule of NADPH. But 1.5 molecules of ATP per NADPH are needed for carbon fixation (see Figure 14-39). To produce extra ATP, the chloroplasts in some species of plants can switch photosystem I into a cyclic mode so that it produces ATP instead of NADPH. In this process, called cyclic photophosphorylation, the high-energy electrons from photosystem I are transferred to the cytochrome b₆-f complex rather than being passed on to NADP⁺. From the b₆-f complex, the electrons are passed back to photosystem I at a low energy. The only net result, besides the conversion of some light energy to heat, is that H⁺ is pumped across the thylakoid membrane by the b₆-f complex as electrons pass through it, thereby increasing the electrochemical proton gradient that drives the ATP synthase. (This is analogous to the right side of the diagram for purple nonsulfur bacteria in Figure 14-71, below.)

To summarize, cyclic photophosphorylation involves only photosystem I, and it produces ATP without the formation of either NADPH or O₂. The relative activities of cyclic and noncyclic electron flows can be regulated by the cell to determine how much light energy is converted into reducing power (NADPH) and how much into high-energy phosphate bonds (ATP).

Photosystems I and II Have Related Structures, and Also Resemble Bacterial Photosystems

The mechanisms of fundamental cell processes such as DNA replication or respiration generally turn out to be the same in eucaryotic cells and in bacteria, even though the number of protein components involved is considerably greater in eucaryotes. Eucaryotes evolved from procaryotes, and the additional proteins presumably were selected for during evolution because they provided an extra degree of efficiency and/or regulation that was useful to the cell.

Photosystems provide a clear example of this type of evolution. Photosystem II, for example, is formed from more than 25 different protein subunits, creating a large assembly in the thylakoid membrane with a mass of about 1 million daltons. The atomic structures of the eucaryotic photosystems are being revealed by a combination of electron and x-ray crystallography. The task is difficult because the complexes are large and embedded in the lipid bilayer. Nevertheless, as illustrated in Figure 14-48, the close relationship of photosystem I, photosystem II, and the photochemical reaction center of purple bacteria has been clearly demonstrated from these atomic-level analyses.

The Proton-Motive Force Is the Same in Mitochondria and Chloroplasts

The presence of the thylakoid space separates a chloroplast into three rather than the two internal compartments of a mitochondrion. The net effect of H⁺ translocation in the two organelles is, however, similar. As illustrated in Figure 14-49, in chloroplasts, H⁺ is pumped out of the stroma (pH 8) into the thylakoid space (pH ~5), creating a gradient of 3–3.5 pH units. This represents a proton-motive force of about 200 mV across the thylakoid membrane, and it drives ATP synthesis by the ATP synthase embedded in this membrane. The force is the same as that across the inner mitochondrial membrane, but nearly all of it is contributed by the pH gradient rather than by a membrane potential, unlike the case in mitochondria.

Like the stroma, the mitochondrial matrix has a pH of about 8. This is created by pumping H⁺ out of the mitochondrion into the cytosol (pH ~7) rather than into an interior space in the organelle. Thus, the pH gradient is relatively small, and most of the proton-motive force across the inner mitochondrial membrane is instead caused by the resulting membrane potential (see Figure 14-13).

For both mitochondria and chloroplasts, the catalytic site of the ATP synthase is at a pH of about 8 and is located in a large organelle compartment (matrix or stroma) that is packed full of soluble enzymes. Consequently, it is here that all of the organelle's ATP is made (see Figure 14-49).

Carrier Proteins in the Chloroplast Inner Membrane Control Metabolite Exchange with the Cytosol

If chloroplasts are isolated in a way that leaves their inner membrane intact, this membrane can be shown to have a selective permeability, reflecting the presence of specific carrier proteins. Most notably, much of the glyceraldehyde 3-phosphate produced by CO₂ fixation in the chloroplast stroma is transported out of the chloroplast by an efficient antiport system that exchanges three-carbon sugar phosphates for an inward flux of inorganic phosphate.

Glyceraldehyde 3-phosphate normally provides the cytosol with an abundant source of carbohydrate, which is used by the cell as the starting point for many other biosyntheses—including the production of sucrose for export. But this is not all that this molecule provides. Once the glyceraldehyde 3-phosphate reaches the cytosol, it is readily converted (by part of the glycolytic pathway) to 1,3-phosphoglycerate and then 3-phosphoglycerate (see p. 97), generating one molecule of ATP and one of NADH. (A similar two-step reaction, but working in reverse, forms glyceraldehyde 3-phosphate in the carbon-fixation cycle; see Figure 14-39.) As a result, the export of glyceraldehyde 3-phosphate from the chloroplast provides not only the main source of fixed carbon to the rest of the cell, but also the reducing power and ATP needed for metabolism outside the chloroplast.

Chloroplasts Also Perform Other Crucial Biosyntheses

The chloroplast performs many biosyntheses in addition to photosynthesis. All of the cell's fatty acids and a number of amino acids, for example, are made by enzymes in the chloroplast stroma. Similarly, the reducing power of light-activated electrons drives the reduction of nitrite (NO₂^-) to ammonia (NH₃) in the chloroplast; this ammonia provides the plant with nitrogen for the synthesis of amino acids and nucleotides. The metabolic importance of the chloroplast for plants and algae therefore extends far beyond its role in photosynthesis.

Summary

Chloroplasts and photosynthetic bacteria obtain high-energy electrons by means of photosystems that capture the electrons that are excited when sunlight is absorbed by chlorophyll molecules. Photosystems are composed of an antenna complex that funnels energy to a photochemical reaction center, where a precisely ordered complex of proteins and pigments allows the energy of an excited electron in chlorophyll to be captured by electron carriers. The best-understood photochemical reaction center is that of purple photosynthetic bacteria, which contain only a single photosystem. In contrast, there are two distinct photosystems in chloroplasts and cyanobacteria. The two photosystems are normally linked in series, and they transfer electrons from water to NADP⁺ to form NADPH, with the concomitant production of a transmembrane electrochemical proton gradient. In these linked photosystems, molecular oxygen (O₂) is generated as a by-product of removing four low-energy electrons from two specifically positioned water molecules.

Compared with mitochondria, chloroplasts have an additional internal membrane (the thylakoid membrane) and a third internal space (the thylakoid space). All electron-transport processes occur in the thylakoid membrane: to make ATP, H⁺ is pumped into the thylakoid space, and a backflow of H⁺ through an ATP synthase then produces the ATP in the chloroplast stroma. This ATP is used in conjunction with the NADPH made by photosynthesis to drive a large number of biosynthetic reactions in the chloroplast stroma, including the all-important carbon-fixation cycle, which creates carbohydrate from CO₂. Along with some other important chloroplast products, this carbohydrate is exported to the cell cytosol, where—as glyceraldehyde 3-phosphate—it provides organic carbon, ATP, and reducing power to the rest of the cell.

Mitochondria and Chloroplasts Contain Complete Genetic Systems

The biosynthesis of mitochondria and plastids requires contributions from two separate genetic systems. Most of the proteins in mitochondria and chloroplasts are encoded by special genes devoted to this purpose in nuclear DNA. These proteins are imported into the organelle from the cytosol after they have been synthesized on cytosolic ribosomes. Other organelle proteins are encoded by organelle DNA and synthesized on ribosomes within the organelle, using organelle-produced mRNA to specify their amino acid sequence (Figure 14-51). The protein traffic between the cytosol and these organelles seems to be unidirectional, as no known proteins are exported from mitochondria or chloroplasts to the cytosol. An exception occurs under special conditions when a cell is about to undergo apoptosis. The release of intermembrane space proteins (including cytochrome c) from mitochondria through the outer mitochondrial membrane is part of a signaling pathway that is triggered in cells undergoing programmed cell death (discussed in Chapter 17).

The processes of organelle DNA transcription, protein synthesis, and DNA replication (Figure 14-52) take place where the genome is located: in the matrix of mitochondria and the stroma of chloroplasts. Although the proteins that mediate these genetic processes are unique to the organelle, most of them are encoded in the nuclear genome. This is all the more surprising because the protein-synthesis machinery of the organelles resembles that of bacteria rather than that of eucaryotes. The resemblance is particularly close in chloroplasts. For example, chloroplast ribosomes are very similar to E. coli ribosomes, both in their structure and in their sensitivity to various antibiotics (such as chloramphenicol, streptomycin, erythromycin, and tetracycline). In addition, protein synthesis in chloroplasts starts with N-formyl methionine, as in bacteria, and not with the methionine used for this purpose in the cytosol of eucaryotic cells. Although mitochondrial genetic systems are much less similar to those of present-day bacteria than are the genetic systems of chloroplasts, their ribosomes are also sensitive to antibacterial antibiotics, and protein synthesis in mitochondria also starts with N-formyl methionine.

Organelle Growth and Division Determine the Number of Mitochondria and Plastids in a Cell

Mitochondria and plastids are large enough to be observed by light microscopy in living cells. For example, mitochondria can be visualized by expressing a genetically engineered fusion of a mitochondrial protein linked to the green fluorescent protein (GFP) in cells, or cells can be incubated with a fluorescent dye that is specifically taken up by mitochondria because of the electrochemical gradient across their membranes. From such images, the mitochondria in living cells are seen to be very dynamic—frequently dividing, fusing, and changing shape (Figure 14-53), as mentioned previously. Division (fission) and fusion of these organelles are topologically complex processes, because the organelles are enclosed by a double membrane and the integrity of the separate mitochondrial compartments must be maintained (Figure 14-54).

The copy number and shape of mitochondria vary dramatically in different cell types and can change in the same cell type under different physiological conditions, ranging from multiple spherical organelles to a single organelle with a branched structure (or reticulum). The arrangement is controlled by the relative rates of mitochondrial division and fusion, which are regulated by dedicated GTPases that reside on mitochondrial membranes. The regulation of mitochondrial morphology and distribution is important for cell differentiation and function. As an example, mutations in Drosophila that impair mitochondrial fusion, and hence cause extensive mitochondrial fragmentation, block sperm development and produce infertility.

There can be many copies of the mitochondrial and plastid genomes in the space enclosed by each organelle's inner membrane. How many of these genomes are present in a single organelle depends on the degree of organelle fragmentation; frequently, many genomes are housed in the same compartment (Table 14-2). In most cells, the replication of the organelle DNA is not limited to the S phase of the cell cycle, when the nuclear DNA replicates, but occurs throughout the cell cycle—out of phase with cell division. Individual organelle DNA molecules seem to be selected at random for replication, so that in a given cell cycle, some may replicate more than once and others not at all. Nonetheless, under constant conditions, the process is regulated to ensure that the total number of organelle DNA molecules doubles in every cell cycle, as required if each cell type is to maintain a constant amount of organelle DNA. When conditions change, the total organelle mass per cell can be regulated according to need. A large increase in mitochondria (as much as five- to tenfold), for example, is observed if a resting skeletal muscle is repeatedly stimulated to contract for a prolonged period.

In special circumstances, organelle division can be precisely controlled by the cell. In some algae that contain only one or a few chloroplasts, the organelle divides just before the cell does, in a plane that is identical to the future plane of cell division.

The Genomes of Mitochondria and Chloroplasts Are Diverse

The multiple copies of mitochondrial and chloroplast DNA contained within the matrix or stroma of these organelles are usually distributed in several clusters, called nucleoids. Nucleoids are thought to be attached to the inner mitochondrial membrane. Although it is not known how the DNA is packaged, the DNA structure in nucleoids is likely to resemble that in bacteria rather than that in eucaryotic chromatin. As in bacteria, for example, there are no histones.

The size range of organelle DNAs is similar to that of viral DNAs. Mitochondrial DNA molecules range in size from less than 6000 nucleotide pairs in Plasmodium falciparum (the human malaria parasite) to more than 300,000 nucleotide pairs in some land plants (Figure 14-55). Like a typical bacterial genome, most mitochondrial DNAs are circular molecules, although linear mitochondrial DNA exists as well. In mammals, the mitochondrial genome is a DNA circle of about 16,500 base pairs (less than 0.001% of the size of the nuclear genome). It is nearly the same size in animals as diverse as Drosophila and sea urchins. The chloroplast genome of land plants ranges in size from 70,000 to 200,000 nucleotide pairs, and it is circular in all organisms examined thus far.

In mammalian cells, mitochondrial DNA makes up less than 1% of the total cellular DNA. In other cells, however, such as the leaves of higher plants or the very large egg cells of amphibians, a much larger fraction of the cellular DNA may be present in mitochondria or chloroplasts (see Table 14-2), and a large fraction of RNA and protein synthesis takes place there.

Mitochondria and Chloroplasts Probably Both Evolved from Endosymbiotic Bacteria

The procaryotic character of the organelle genetic systems, especially striking in chloroplasts, suggests that mitochondria and chloroplasts evolved from bacteria that were endocytosed more than 1 billion years ago. According to this endosymbiont hypothesis, eucaryotic cells started out as anaerobic organisms without mitochondria or chloroplasts and then established a stable endosymbiotic relation with a bacterium, whose oxidative phosphorylation system they subverted for their own use (Figure 14-56). The endocytic event that led to the development of mitochondria is presumed to have occurred when oxygen entered the atmosphere in substantial amounts, about 1.5 × 10⁹ years ago, before animals and plants separated (see Figure 14-69).

Plant and algal chloroplasts seem to have been derived later from an endocytic event involving an oxygen-producing photosynthetic bacterium. To explain the different pigments and properties of the chloroplasts found in present-day higher plants and algae, it is usually assumed that at least three independent endosymbiotic events occurred.

Most of the genes encoding present-day mitochondrial and chloroplast proteins are in the cell nucleus. Thus, an extensive transfer of genes from organelle to nuclear DNA must have occurred during eucaryote evolution. In contrast, present organelle genomes are stable, indicating that a successful transfer is a rare evolutionary process. This is expected, because a gene moved from organelle DNA needs to change to become a functional nuclear gene: it must adapt to the nuclear and cytoplasmic transcription and translation requirements, and also acquire a signal sequence so that the encoded protein can be delivered to the organelle after its synthesis in the cytosol.

The gene transfer hypothesis explains why many of the nuclear genes encoding mitochondrial and chloroplast proteins resemble bacterial genes. The amino acid sequence of the chicken mitochondrial enzyme superoxide dismutase, for example, resembles the corresponding bacterial enzyme much more than it resembles the superoxide dismutase found in the cytosol of the same eucaryotic cell. Further evidence that such DNA transfers have occurred during evolution comes from the discovery of some noncoding DNA sequences in nuclear DNA that seem to be of recent mitochondrial origin; they have apparently integrated into the nuclear genome as “junk DNA.”

Gene transfer seems to have been a gradual process. When mitochondrial genomes encoding different numbers of proteins are compared, a pattern of sequential reduction of encoded mitochondrial functions emerges (Figure 14-57). The smallest and presumably most highly evolved mitochondrial genomes, for example, encode only a few inner-membrane proteins involved in electron-transport reactions, plus ribosomal RNAs and tRNAs. Mitochondrial genomes that have remained more complex contain this same subset of genes, plus others. The most complex genomes are characterized by the presence of many extra genes compared with animal and yeast mitochondrial genomes. Many of these genes encode components of the mitochondrial genetic system, such as RNA polymerase subunits and ribosomal proteins; these genes are instead found in the cell nucleus in organisms that have reduced their mitochondrial DNA content.

What type of bacterium gave rise to the mitochondrion? From sequence comparisons, it seems that mitochondria are descendants of a particular type of purple photosynthetic bacterium that had previously lost its ability to perform photosynthesis and was left with only a respiratory chain. It is not certain that all mitochondria have originated from the same endosymbiotic event, however.

Mitochondrial Genomes Have Several Surprising Features

The relatively small size of the human mitochondrial genome made it a particularly attractive target for early DNA-sequencing projects, and in 1981, the complete sequence of its 16,569 nucleotides was published. By comparing this sequence with known mitochondrial tRNA sequences and with the partial amino acid sequences available for proteins encoded by the mitochondrial DNA, all of the human mitochondrial genes were mapped on the circular DNA molecule (Figure 14-58).

Compared with nuclear, chloroplast, and bacterial genomes, the human mitochondrial genome has several surprising features:

• 1

  Dense gene packing. Unlike other genomes, nearly every nucleotide seems to be part of a coding sequence, either for a protein or for one of the rRNAs or tRNAs. Since these coding sequences run directly into each other, there is very little room left for regulatory DNA sequences.

• 2

  Relaxed codon usage. Whereas 30 or more tRNAs specify amino acids in the cytosol and in chloroplasts, only 22 tRNAs are required for mitochondrial protein synthesis. The normal codon-anticodon pairing rules are relaxed in mitochondria, so that many tRNA molecules recognize any one of the four nucleotides in the third (wobble) position. Such “2 out of 3” pairing allows one tRNA to pair with any one of four codons and permits protein synthesis with fewer tRNA molecules.

• 3
  Variant genetic code. Perhaps most surprising, comparisons of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicate that the genetic code is different: 4 of the 64 codons have different “meanings” from those of the same codons in other genomes (Table 14-3).

The observation that the genetic code is nearly the same in all organisms provides strong evidence that all cells have evolved from a common ancestor. How, then, does one explain the few differences in the genetic code in many mitochondria? A hint comes from the finding that the mitochondrial genetic code is different in different organisms. In the mitochondrion with the largest number of genes in Figure 14-57, that of the protozoan Reclinomonas, the genetic code is unchanged from the standard genetic code of the cell nucleus. Yet UGA, which is a stop codon elsewhere, is read as tryptophan in mitochondria of mammals, fungi, and invertebrates. Similarly, the codon AGG normally codes for arginine, but it codes for stop in the mitochondria of mammals and codes for serine in the mitochondria of Drosophila (see Table 14-3). Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes an occasional change in the meaning of a rare codon tolerable, whereas such a change in a large genome would alter the function of many proteins and thereby destroy the cell.

Animal Mitochondria Contain the Simplest Genetic Systems Known

Comparisons of DNA sequences in different organisms reveal that the rate of nucleotide substitution during evolution has been 10 times greater in mitochondrial genomes than in nuclear genomes, which presumably is due to a reduced fidelity of mitochondrial DNA replication, inefficient DNA repair, or both. Because only about 16,500 DNA nucleotides need to be replicated and expressed as RNAs and proteins in animal cell mitochondria, the error rate per nucleotide copied by DNA replication, maintained by DNA repair, transcribed by RNA polymerase, or translated into protein by mitochondrial ribosomes can be relatively high without damaging one of the relatively few gene products. This could explain why the mechanisms that perform these processes are relatively simple compared with those used for the same purpose elsewhere in cells. The presence of only 22 tRNAs and the unusually small size of the rRNAs (less than two-thirds the size of the E. coli rRNAs), for example, would be expected to reduce the fidelity of protein synthesis in mitochondria, although this has not yet been tested adequately.

The relatively high rate of evolution of mitochondrial genes makes a comparison of mitochondrial DNA sequences especially useful for estimating the dates of relatively recent evolutionary events, such as the steps in primate evolution.

Some Organelle Genes Contain Introns

The processing of precursor RNAs has an important role in the two mitochondrial systems studied in most detail—human and yeast. In human cells, both strands of the mitochondrial DNA are transcribed at the same rate from a single promoter region on each strand, producing two different giant RNA molecules, each containing a full-length copy of one DNA strand. Transcription is therefore completely symmetric. The transcripts made on one strand are extensively processed by nuclease cleavage to yield the two rRNAs, most of the tRNAs, and about 10 poly-A-containing RNAs. In contrast, the transcript of the other strand is processed to produce only 8 tRNAs and 1 small poly-A-containing RNA; the remaining 90% of this transcript apparently contains no useful information (being complementary to coding sequences synthesized on the other strand) and is degraded. The poly-A-containing RNAs are the mitochondrial mRNAs: although they lack a cap structure at their 5′ end, they carry a poly-A tail at their 3′ end that is added posttranscriptionally by a mitochondrial poly-A polymerase.

Unlike human mitochondrial genes, some plant and fungal (including yeast) mitochondrial genes contain introns, which must be removed by RNA splicing. Introns have also been found in plant chloroplast genes. Many of the introns in organelle genes consist of a family of related nucleotide sequences that are capable of splicing themselves out of the RNA transcripts by RNA-mediated catalysis (discussed in Chapter 6), although these self-splicing reactions are generally aided by proteins. The presence of introns in organelle genes is surprising, as introns are not common in the genes of the bacteria whose ancestors are thought to have given rise to mitochondria and plant chloroplasts.

In yeasts, the same mitochondrial gene may have an intron in one strain but not in another. Such “optional introns” seem to be able to move in and out of genomes like transposable elements. In contrast, introns in other yeast mitochondrial genes have also been found in a corresponding position in the mitochondria of Aspergillus and Neurospora, implying that they were inherited from a common ancestor of these three fungi. It is possible that these intron sequences are of ancient origin—tracing back to a bacterial ancestor—and that, although they have been lost from many bacteria, they have been preferentially retained in some organelle genomes where RNA splicing is regulated to help control gene expression.

The Chloroplast Genome of Higher Plants Contains About 120 Genes

More than 20 chloroplast genomes have now been sequenced. The genomes of even distantly related plants (such as tobacco and liverwort) are nearly identical, and even those of green algae are closely related (Figure 14-59). Chloroplast genes are involved in four main types of processes: transcription, translation, photosynthesis, and the biosynthesis of small molecules such as amino acids, fatty acids, and pigments. Plant chloroplast genes also encode at least 40 proteins whose functions are as yet unknown; in addition, about twice that many genes of unknown function are present in the chloroplasts of some algae. Paradoxically, all of the known proteins encoded in the chloroplast are part of larger protein complexes that also contain one or more subunits encoded in the nucleus. We discuss possible reasons for this paradox later.

The similarities between the genomes of chloroplasts and bacteria are striking. The basic regulatory sequences, such as transcription promoters and terminators, are virtually identical in the two cases. The amino acid sequences of the proteins encoded in chloroplasts are clearly recognizable as bacterial, and several clusters of genes with related functions (such as those encoding ribosomal proteins) are organized in the same way in the genomes of chloroplasts, E. coli, and cyanobacteria.

Further comparisons of large numbers of homologous nucleotide sequences should help clarify the exact evolutionary pathway from bacteria to chloroplasts, but several conclusions can already be drawn:

• 1

  Chloroplasts in higher plants arose from photosynthetic bacteria.

• 2

  The chloroplast genome has been stably maintained for at least several hundred million years, the estimated time of divergence of liverwort and tobacco.

• 3

  Many of the genes of the original bacterium are now present in the nuclear genome, where they have been integrated and are stably maintained. In higher plants, for example, two-thirds of the 60 or so chloroplast ribosomal proteins are encoded in the cell nucleus; these genes have a clear bacterial ancestry, and the chloroplast ribosomes retain their original bacterial properties.

Mitochondrial Genes Are Inherited by a Non-Mendelian Mechanism

Many experiments on the mechanisms of mitochondrial biogenesis have been performed with Saccharomyces cerevisiae (baker's yeast). There are several reasons for this preference. First, when grown on glucose, this yeast has an ability to live by glycolysis alone and can therefore survive with defective mitochondria that cannot perform oxidative phosphorylation. This makes it possible to grow cells with mutations in mitochondrial or nuclear DNA that interfere with mitochondrial function; such mutations are lethal in many other eucaryotes. Second, yeasts are simple unicellular eucaryotes that are easy to grow and characterize biochemically. Finally, these yeast cells normally reproduce asexually by budding, but they can also reproduce sexually. During sexual reproduction two haploid cells mate and fuse to form a diploid zygote, which can either grow mitotically or divide by meiosis to produce new haploid cells.

The ability to control the alternation between asexual and sexual reproduction in the laboratory greatly facilitates genetic analyses. Mutations in mitochondrial genes are not inherited in accordance with the Mendelian rules that govern the inheritance of nuclear genes. Therefore, long before the mitochondrial genome could be sequenced, genetic studies revealed which of the genes involved in yeast mitochondrial function are located in the nucleus and which in the mitochondria. An example of non-Mendelian (cytoplasmic) inheritance of mitochondrial genes in a haploid yeast cell is shown in Figure 14-60. In this example, we follow the inheritance of a mutant gene that makes mitochondrial protein synthesis resistant to chloramphenicol.

When a chloramphenicol-resistant haploid cell mates with a chloramphenicol-sensitive wild-type haploid cell, the resulting diploid zygote contains a mixture of mutant and wild-type genomes. The two mitochondrial networks fuse in the zygote, creating one continuous reticulum that contains genomes of both parental cells. When the zygote undergoes mitosis, copies of both mutant and wild-type mitochondrial DNA are segregated to the diploid daughter cell. In the case of nuclear DNA, each daughter cell receives exactly two copies of each chromosome, one from each parent. By contrast, in the case of mitochondrial DNA, the daughter cell may inherit either more copies of the mutant DNA or more copies of the wild-type DNA. Successive mitotic divisions can further enrich for either DNA, so that subsequently many cells will arise that contain mitochondrial DNA of only one genotype. This stochastic process is called mitotic segregation.

When diploid cells that have segregated their mitochondrial genomes in this way undergo meiosis to form four haploid daughter cells, each of the four daughters receives the same mitochondrial genes. This type of inheritance is called non-Mendelian, or cytoplasmic inheritance, to contrast it with the Mendelian inheritance of nuclear genes (see Figure 14-60). When non-Mendelian inheritance occurs, it demonstrates that the gene in question is located outside the nuclear chromosomes.

Although clusters of mitochondrial DNA molecules (nucleoids) are relatively immobile in the mitochondrial reticulum because of their anchorage to the inner membrane, individual nucleoids occasionally come together. This occurs frequently, for example, at sites where the two parental mitochondrial networks fuse during zygote formation. When different DNAs are present in the same nucleoid, genetic recombination can occur. This recombination can result in mitochondrial genomes that contain DNA from both parent cells, which are stably inherited after their mitotic segregation.

Organelle Genes Are Maternally Inherited in Many Organisms

The consequences of cytoplasmic inheritance are more profound for some organisms, including ourselves, than they are for yeasts. In yeasts, when two haploid cells mate, they are equal in size and contribute equal amounts of mitochondrial DNA to the zygote (see Figure 14-60). Mitochondrial inheritance in yeasts is therefore biparental: both parents contribute equally to the mitochondrial gene pool of the progeny (although, as we have just seen, after several generations of vegetative growth, the individual progeny often contain mitochondria from only one parent). In higher animals, by contrast, the egg cell always contributes much more cytoplasm to the zygote than does the sperm. One would therefore expect mitochondrial inheritance in higher animals to be nearly uniparental—or, more precisely, maternal. Such maternal inheritance has been demonstrated in laboratory animals. When animals carrying type A mitochondrial DNA are crossed with animals carrying type B, the progeny contain only the maternal type of mitochondrial DNA. Similarly, by following the distribution of variant mitochondrial DNA sequences in large families, it has been shown that human mitochondrial DNA is maternally inherited.

In about two-thirds of higher plants, the chloroplasts from the male parent (contained in pollen grains) do not enter the zygote, so that chloroplast as well as mitochondrial DNA is maternally inherited. In other plants, the pollen chloroplasts enter the zygote, making chloroplast inheritance biparental. In such plants, defective chloroplasts are a cause of variegation: a mixture of normal and defective chloroplasts in a zygote may sort out by mitotic segregation during plant growth and development, thereby producing alternating green and white patches in leaves. The green patches contain normal chloroplasts, while the white patches contain defective chloroplasts (Figure 14-61).

A fertilized human egg carries perhaps 2000 copies of the human mitochondrial genome, all but one or two inherited from the mother. A human in which all of these genomes carried a deleterious mutation would generally not survive. But some mothers carry a mixed population of both mutant and normal mitochondrial genomes. Their daughters and sons inherit this mixture of normal and mutant mitochondrial DNAs and are healthy unless the process of mitotic segregation by chance results in a majority of defective mitochondria in a particular tissue. Muscle and nervous tissues are most at risk, because of their need for particularly large amounts of ATP.

An inherited disease in humans caused by a mutation in mitochondrial DNA can be recognized by its passage from affected mothers to both their daughters and their sons, with the daughters but not the sons producing grandchildren with the disease. As expected from the random nature of mitotic segregation, the symptoms of these diseases vary greatly between different family members—including not only the severity and age of onset, but also which tissue is affected.

Consider, for example, the inherited disease myoclonic epilepsy and ragged red fiber disease (MERRF), which can be caused by a mutation in one of the mitochondrial transfer RNA genes. This disease appears when, by chance, a particular tissue inherits a threshold amount of defective mitochondrial DNA genomes. Above this threshold, the pool of defective tRNA causes a decrease in the synthesis of the mitochondrial proteins required for electron transport and production of ATP. The result may be muscle weakness or heart problems (from effects on heart muscle), forms of epilepsy or dementia (from effects on nerve cells), or other symptoms. Not surprisingly, a similar variability in phenotypes is found for many other mitochondrial diseases.

Because of the unusually high rate of mutation observed in mitochondria, it has also been suggested that mutations that accumulate in mitochondrial DNAs may contribute to many of the medical problems of old age.

Petite Mutants in Yeasts Demonstrate the Overwhelming Importance of the Cell Nucleus for Mitochondrial Biogenesis

Genetic studies of yeasts have had a crucial role in the analysis of mitochondrial biogenesis. A striking example is provided by studies of yeast mutants that contain large deletions in their mitochondrial DNA, so that all mitochondrial protein synthesis is abolished. Not surprisingly, these mutants cannot make respiring mitochondria. Some of these mutants lack mitochondrial DNA altogether. Because they form unusually small colonies when grown in media with low glucose, all mutants with such defective mitochondria are called cytoplasmic petite mutants.

Although petite mutants cannot synthesize proteins in their mitochondria and therefore cannot make mitochondria that produce ATP, they nevertheless contain mitochondria. These mitochondria have a normal outer membrane and an inner membrane with poorly developed cristae (Figure 14-62). They contain virtually all the mitochondrial proteins that are specified by nuclear genes and imported from the cytosol—including DNA and RNA polymerases, all of the citric acid cycle enzymes, and most inner membrane proteins—demonstrating the overwhelming importance of the nucleus in mitochondrial biogenesis. Petite mutants also show that an organelle that divides by fission can replicate indefinitely in the cytoplasm of proliferating eucaryotic cells, even in the complete absence of its own genome. It is possible that peroxisomes normally replicate in this way (see Figure 12-34).

For chloroplasts, the nearest equivalent to yeast mitochondrial petite mutants are mutants of unicellular algae such as Euglena. Mutant algae in which no chloroplast protein synthesis occurs still contain chloroplasts and are perfectly viable if oxidizable substrates are provided. If the development of mature chloroplasts is blocked in higher plants, however, either by raising the plants in the dark or because chloroplast DNA is defective or absent, the plants die.

Mitochondria and Plastids Contain Tissue-specific Proteins that Are Encoded in the Cell Nucleus

Mitochondria can have specialized functions in particular types of cells. The urea cycle, for example, is the central metabolic pathway in mammals for disposing of cellular breakdown products that contain nitrogen. These products are excreted in the urine as urea. Nucleus-encoded enzymes in the mitochondrial matrix perform several steps in the cycle. Urea synthesis occurs in only a few tissues, such as the liver, and the required enzymes are synthesized and imported into mitochondria only in these tissues.

The respiratory enzyme complexes in the mitochondrial inner membrane of mammals contain several tissue-specific, nucleus-encoded subunits that are thought to act as regulators of electron transport. Thus, some humans with a genetic muscle disease have a defective subunit of cytochrome oxidase; since the subunit is specific to skeletal muscle cells, their other cells, including their heart muscle cells, function normally, allowing the individuals to survive. As would be expected, tissue-specific differences are also found among the nucleus-encoded proteins in chloroplasts.

Mitochondria Import Most of Their Lipids; Chloroplasts Make Most of Theirs

The biosynthesis of new mitochondria and chloroplasts requires lipids in addition to nucleic acids and proteins. Chloroplasts tend to make the lipids they require. In spinach leaves, for example, all cellular fatty acid synthesis takes place in the chloroplast, although desaturation of the fatty acids occurs elsewhere. The major glycolipids of the chloroplast are also synthesized locally.

Mitochondria, in contrast, import most of their lipids. In animal cells, the phospholipids phosphatidylcholine and phosphatidylserine are synthesized in the endoplasmic reticulum and then transferred to the outer membrane of mitochondria. In addition to decarboxylating imported phosphatidylserine to phosphatidylethanolamine, the main reaction of lipid biosynthesis catalyzed by the mitochondria themselves is the conversion of imported lipids to cardiolipin (bisphosphatidylglycerol). Cardiolipin is a “double” phospholipid that contains four fatty acid tails (Figure 14-63). It is found mainly in the mitochondrial inner membrane, where it constitutes about 20% of the total lipid.

We discuss the important question of how specific cytosolic proteins are imported into mitochondria and chloroplasts in Chapter 12.

Why Do Mitochondria and Chloroplasts Have Their Own Genetic Systems?

Why do mitochondria and chloroplasts require their own separate genetic systems, when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? The question is not trivial, because maintaining a separate genetic system is costly: more than 90 proteins—including many ribosomal proteins, aminoacyl-tRNA synthases, DNA and RNA polymerases, and RNA-processing and RNA-modifying enzymes—must be encoded by nuclear genes specifically for this purpose (Figure 14-64). The amino acid sequences of most of these proteins in mitochondria and chloroplasts differ from those of their counterparts in the nucleus and cytosol, and it appears that these organelles have relatively few proteins in common with the rest of the cell. This means that the nucleus must provide at least 90 genes just to maintain each organelle's genetic system. The reason for such a costly arrangement is not clear, and the hope that the nucleotide sequences of mitochondrial and chloroplast genomes would provide the answer has proved to be unfounded. We cannot think of compelling reasons why the proteins made in mitochondria and chloroplasts should be made there rather than in the cytosol.

At one time, it was suggested that some proteins have to be made in the organelle because they are too hydrophobic to get to their site in the membrane from the cytosol. More recent studies, however, make this explanation implausible. In many cases, even highly hydrophobic subunits are synthesized in the cytosol. Moreover, although the individual protein subunits in the various mitochondrial enzyme complexes are highly conserved in evolution, their site of synthesis is not (see Figure 14-57). The diversity in the location of the genes coding for the subunits of functionally equivalent proteins in different organisms is difficult to explain by any hypothesis that postulates a specific evolutionary advantage of present-day mitochondrial or chloroplast genetic systems.

Perhaps the organelle genetic systems are an evolutionary dead-end. In terms of the endosymbiont hypothesis, this would mean that the process whereby the endosymbionts transferred most of their genes to the nucleus stopped before it was complete. Further transfers may have been ruled out, for mitochondria, by recent alterations in the mitochondrial genetic code that made the remaining mitochondrial genes nonfunctional if they were transferred to the nucleus.

Summary

Mitochondria and chloroplasts grow in a coordinated process that requires the contribution of two separate genetic systems—one in the organelle and one in the cell nucleus. Most of the proteins in these organelles are encoded by nuclear DNA, synthesized in the cytosol, and then imported individually into the organelle. Some organelle proteins and RNAs are encoded by the organelle DNA and are synthesized in the organelle itself. The human mitochondrial genome contains about 16,500 nucleotides and encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 different polypeptide chains. Chloroplast genomes are about 10 times larger and contain about 120 genes. But partly functional organelles form in normal numbers even in mutants that lack a functional organelle genome, demonstrating the overwhelming importance of the nucleus for the biogenesis of both organelles.

The ribosomes of chloroplasts closely resemble bacterial ribosomes, while mitochondrial ribosomes show both similarities and differences that make their origin more difficult to trace. Protein similarities, however, suggest that both organelles originated when a primitive eucaryotic cell entered into a stable endosymbiotic relationship with a bacterium. A purple bacterium is thought to have given rise to the mitochondrion, and (later) a relative of a cyanobacterium is thought to have given rise to the plant chloroplast. Although many of the genes of these ancient bacteria still function to make organelle proteins, most of them have become integrated into the nuclear genome, where they encode bacterialike enzymes that are synthesized on cytosolic ribosomes and then imported into the organelle.

The Earliest Cells Probably Produced ATP by Fermentation

As explained in Chapter 1, the first living cells on Earth are thought to have arisen more than 3.5 × 10⁹ years ago, when the Earth was not more than about 10⁹ years old. The environment lacked oxygen but was presumably rich in geochemically produced organic molecules, and some of the earliest metabolic pathways for producing ATP may have resembled present-day forms of fermentation.

In the process of fermentation, ATP is made by a phosphorylation event that harnesses the energy released when a hydrogen-rich organic molecule, such as glucose, is partly oxidized (see Figure 2-72). The electrons lost from the oxidized organic molecules are transferred (via NADH or NADPH) to a different organic molecule (or to a different part of the same molecule), which thereby becomes more reduced. At the end of the fermentation process, one or more of the organic molecules produced are excreted into the medium as metabolic waste products; others, such as pyruvate, are retained by the cell for biosynthesis.

The excreted end-products are different in different organisms, but they tend to be organic acids (carbon compounds that carry a COOH group). Among the most important of such products in bacterial cells are lactic acid (which also accumulates in anaerobic mammalian glycolysis) and formic, acetic, propionic, butyric, and succinic acids.

Electron-transport Chains Enabled Anaerobic Bacteria to Use Nonfermentable Molecules as Their Major Source of Energy

The early fermentation processes would have provided not only the ATP but also the reducing power (as NADH or NADPH) required for essential biosyntheses. Thus, many of the major metabolic pathways could have evolved while fermentation was the only mode of energy production. With time, however, the metabolic activities of these procaryotic organisms must have changed the local environment, forcing organisms to evolve new biochemical pathways. The accumulation of waste products of fermentation, for example, might have resulted in the following series of changes:

• Stage 1. The continuous excretion of organic acids lowered the pH of the environment, favoring the evolution of proteins that function as transmembrane H⁺ pumps that can pump H⁺ out of the cell to protect it from the dangerous effects of intracellular acidification. One of these pumps may have used the energy available from ATP hydrolysis and could have been the ancestor of the present-day ATP synthase.

• Stage 2. At the same time as nonfermentable organic acids were accumulating in the environment and favoring the evolution of an ATP-consuming H⁺ pump, the supply of geochemically generated fermentable nutrients, which provided the energy for the pumps and for all other cellular processes, was dwindling. This favored bacteria that could excrete H⁺ without hydrolyzing ATP, allowing the ATP to be conserved for other cellular activities. Selective pressures of this kind might have led to the first membrane-bound proteins that could use electron transport between molecules of different redox potentials as the energy source for transporting H⁺ across the plasma membrane. Some of these proteins would have found their electron donors and electron acceptors among the nonfermentable organic acids that had accumulated. Many such electron-transport proteins can be found in present-day bacteria; some bacteria that grow on formic acid, for example, pump H⁺ by using the small amount of redox energy derived from the transfer of electrons from formic acid to fumarate (Figure 14-65). Others have similar electron-transport components devoted solely to the oxidation and reduction of inorganic substrates (see Figure 14-67, for example).

• Stage 3. Eventually some bacteria developed H⁺-pumping electron-transport systems that were efficient enough to harness more redox energy than they needed just to maintain their internal pH. Now, bacteria that carried both types of H⁺ pumps were at an advantage. In these cells, a large electrochemical proton gradient generated by excessive H⁺ pumping allowed protons to leak back into the cell through the ATP-driven H⁺ pumps, thereby running them in reverse, so that they functioned as ATP synthases to make ATP. Because such bacteria required much less of the increasingly scarce supply of fermentable nutrients, they proliferated at the expense of their neighbors.

These three hypothetical stages in the evolution of oxidative phosphorylation mechanisms are summarized in Figure 14-66.

By Providing an Inexhaustible Source of Reducing Power, Photosynthetic Bacteria Overcame a Major Evolutionary Obstacle

The evolutionary steps just outlined would have solved the problem of maintaining both a neutral intracellular pH and an abundant store of energy, but these steps would not have solved another problem that was equally serious. The depletion of organic nutrients from the environment meant that organisms had to find some alternative source of carbon to make the sugars that served as the precursors for so many other cellular molecules. Although the CO₂ in the atmosphere provided an abundant potential carbon source, to convert it into an organic molecule such as a carbohydrate requires that the fixed CO₂ be reduced by a strong electron donor, such as NADH or NADPH, which can provide the high-energy electrons needed to generate each (CH₂O) unit from CO₂ (see Figure 14-39). Early in cellular evolution, strong reducing agents (electron donors) would have been plentiful as products of fermentation. But as the supply of fermentable nutrients dwindled and a membrane-bound ATP synthase began to produce most of the ATP, the plentiful supply of NADH and other reducing agents would have disappeared. It thus became imperative for cells to evolve a new way of generating strong reducing agents.

Presumably, the main reducing agents still available were the organic acids produced by the anaerobic metabolism of carbohydrates, inorganic molecules such as hydrogen sulfide (H₂S) generated geochemically, and water. But the reducing power of these molecules is far too weak to be useful for CO₂ fixation. An early supply of strong electron donors could have been generated by using the electrochemical proton gradient across the plasma membrane to drive a reverse electron flow. This would have required the evolution of membrane-bound enzyme complexes resembling an NADH dehydrogenase, and mechanisms of this kind survive in the anaerobic metabolism of some present-day bacteria (Figure 14-67).

The major evolutionary breakthrough in energy metabolism, however, was almost certainly the development of photochemical reaction centers that could use the energy of sunlight to produce molecules such as NADH. It is thought that this occurred early in the process of cellular evolution—more than 3 × 10⁹ years ago, in the ancestors of the green sulfur bacteria. Present-day green sulfur bacteria use light energy to transfer hydrogen atoms (as an electron plus a proton) from H₂S to NADPH, thereby creating the strong reducing power required for carbon fixation (Figure 14-68). Because the electrons removed from H₂S are at a much more negative redox potential than those of H₂O (-230 mV for H₂S compared +820 mV for H₂O), one quantum of light absorbed by the single photosystem in these bacteria is sufficient to achieve a high enough redox potential to generate NADPH via a relatively simple photosynthetic electron-transport chain.

The Photosynthetic Electron-transport Chains of Cyanobacteria Produced Atmospheric Oxygen and Permitted New Life-Forms

The next step, which is thought to have occurred with the development of the cyanobacteria at least 3 × 10⁹ years ago, was the evolution of organisms capable of using water as the electron source for CO₂ reduction. This entailed the evolution of a water-splitting enzyme and also required the addition of a second photosystem, acting in series with the first, to bridge the enormous gap in redox potential between H₂O and NADPH. Present-day structural homologies between photosystems suggest that this change involved the cooperation of a photosystem derived from green bacteria (photosystem I) with a photosystem derived from purple bacteria (photosystem II). The biological consequences of this evolutionary step were far-reaching. For the first time, there were organisms that made only very minimal chemical demands on their environment. These cells could spread and evolve in ways denied to the earlier photosynthetic bacteria, which needed H₂S or organic acids as a source of electrons. Consequently, large amounts of biologically synthesized, reduced organic materials accumulated. Moreover, oxygen entered the atmosphere for the first time.

Oxygen is highly toxic because the oxidation reactions it brings about can randomly alter biological molecules. Many present-day anaerobic bacteria, for example, are rapidly killed when exposed to air. Thus, organisms on the primitive Earth would have had to evolve protective mechanisms against the rising O₂ levels in the environment. Late evolutionary arrivals, such as ourselves, have numerous detoxifying mechanisms that protect our cells from the ill effects of oxygen. Even so, an accumulation of oxidative damage to our macromolecules is postulated to be a major cause of human aging.

The increase in atmospheric O₂ was very slow at first and would have allowed a gradual evolution of protective devices. The early seas contained large amounts of iron in its ferrous oxidation state (Fe²⁺), and nearly all the O₂ produced by early photosynthetic bacteria was utilized in converting Fe²⁺ to Fe³⁺. This conversion caused the precipitation of huge amounts of ferric oxides, and the extensive banded iron formations in sedimentary rocks beginning about 2.7 × 10⁹ years ago help to date the spread of the cyanobacteria. By about 2 × 10⁹ years ago, the supply of ferrous iron was exhausted, and the deposition of further iron precipitates ceased. Geological evidence suggests that O₂ levels in the atmosphere then began to rise, reaching current levels between 0.5 and 1.5 × 10⁹ years ago (Figure 14-69).

The availability of O₂ made possible the development of bacteria that relied on aerobic metabolism to make their ATP. As explained previously, these organisms could harness the large amount of energy released by breaking down carbohydrates and other reduced organic molecules all the way to CO₂ and H₂O. Components of preexisting electron-transport complexes were modified to produce a cytochrome oxidase, so that the electrons obtained from organic or inorganic substrates could be transported to O₂ as the terminal electron acceptor. Depending on the availability of light and O₂, many present-day purple photosynthetic bacteria can switch between photosynthesis and respiration, requiring only relatively minor reorganizations of their electron-transport chains.

As organic materials accumulated on Earth as a result of photosynthesis, some photosynthetic bacteria (including the precursors of E. coli) lost their ability to survive on light energy alone and came to rely entirely on respiration. As described previously (see Figure 14-56), it has been suggested that mitochondria first arose some 1.5 × 10⁹ years ago, when a primitive eucaryotic cell endocytosed such a respiration-dependent bacterium. Plants are believed to have evolved somewhat later, when a descendant of this early aerobic eucaryotic cell endocytosed a photosynthetic bacterium that became the precursor of chloroplasts. Present-day chloroplasts are so different in some types of algae that chloroplasts probably evolved separately in different algal lineages. Figure 14-70 relates these postulated pathways to the various types of bacteria discussed in this chapter.

Evolution is always conservative, taking parts of the old and building on them to create something new. Thus, parts of the electron-transport chains that were derived to service anaerobic bacteria 3–4 × 10⁹ years ago probably survive, in altered form, in the mitochondria and chloroplasts of today's higher eucaryotes. Consider, for example, the striking homology in structure and function between the enzyme complex that pumps H⁺ in the central segment of the mitochondrial respiratory chain (the cytochrome b-c₁ complex) and its analogs in the electron-transport chains of both bacteria and chloroplasts (Figure 14-71).

Summary

Early cells are believed to have been bacteriumlike organisms living in an environment rich in highly reduced organic molecules that had been formed by geochemical processes over the course of hundreds of millions of years. They may have derived most of their ATP by converting these reduced organic molecules to a variety of organic acids, which were then released as waste products. By acidifying the environment, these fermentations may have led to the evolution of the first membrane-bound H⁺ pumps, which could maintain a neutral pH in the cell interior by pumping out H⁺. The properties of present-day bacteria suggest that an electron-transport-driven H⁺ pump and an ATP-driven H⁺ pump first arose in this anaerobic environment. Reversal of the ATP-driven pump would have allowed it to function as an ATP synthase. As more effective electron-transport chains developed, the energy released by redox reactions between inorganic molecules and/or accumulated nonfermentable compounds produced a large electrochemical proton gradient, which could be harnessed by the ATP-driven pump for ATP production.

Because preformed organic molecules were replenished only very slowly by geochemical processes, the proliferation of bacteria that used them as the source of both carbon and reducing power could not go on forever. The depletion of fermentable organic nutrients presumably led to the evolution of bacteria that could use CO₂to make carbohydrates. By combining parts of the electron-transport chains that had developed earlier, light energy was harvested by a single photosystem in photosynthetic bacteria to generate the NADPH required for carbon fixation. The subsequent appearance of the more complex photosynthetic electron-transport chains of the cyanobacteria allowed H₂O to be used as the electron donor for NADPH formation, rather than the much less abundant electron donors required by other photosynthetic bacteria. Life could then proliferate over large areas of the Earth, so that reduced organic molecules accumulated again.

About 2×10⁹ years ago, the O₂released by photosynthesis in cyanobacteria began to accumulate in the atmosphere. Once both organic molecules and O₂had become abundant, electron-transport chains became adapted for the transport of electrons from NADH to O₂, and efficient aerobic metabolism developed in many bacteria. Exactly the same aerobic mechanisms operate today in the mitochondria of eucaryotes, and there is increasing evidence that both mitochondria and chloroplasts evolved from aerobic bacteria that were endocytosed by primitive eucaryotic cells.