Mitochondria, which are present in virtually all eucaryotic cells, and plastids (most notably chloroplasts), which occur only in plants, are membrane-bounded organelles that convert energy to forms that can be used to drive cellular reactions. Consistent with their importance in metabolism, they generally occupy a major fraction of the total cell volume. In electron micrographs the most striking morphological feature of mitochondria and chloroplasts is the large amount of internal membrane they contain. As we shall see, this membrane has a crucial role in the function of these energy-converting organelles by providing a framework for electron-transport processes.
Although mitochondria convert energy derived from chemical fuels whereas chloroplasts convert energy derived from sunlight, the two types of organelles are organized similarly; moreover, both produce large amounts of ATP by the same mechanism. This striking conclusion emerged from painstaking studies carried out over the past 30 years.
Energy from sunlight or the oxidation of foodstuffs is first used to create an electrochemical proton gradient across a membrane. This gradient serves as a versatile energy store and is used to drive a variety of reactions in mitochondria, chloroplasts, and bacteria.
).
Foremost among these
machines is the enzyme ATP synthase, which uses the energy of the
H+ flow to synthesize ATP from ADP and
Pi. Other proteins couple the
H+ flow to the transport of specific metabolites into and
out of the organelles. In bacteria the
electrochemical proton gradient itself is as important a store of directly
usable
energy as is the ATP it generates: the gradient not only drives many transport
processes, it also drives the rapid rotation of the bacterial flagellum,
which allows the
bacterium to swim.
Inputs are light green,products are blue,and the path of electron flow is indicated by red arrows. Note that the electron-motive force generated by the two chloroplast photosystems enables the chloroplast (B) to drive electron transfer from H2O to carbohydrate, which is opposite to the direction of electron transfer in the mitochondrion (A).
. The entire set
of proteins and small molecules involved in this orderly sequence of
electron transfers within the membrane is called an
electron-transport chain.
.
It is generally believed that the energy-converting organelles of eucaryotes evolved from procaryotes that were engulfed by primitive eucaryotic cells and developed a symbiotic relationship with them about 1.5 x 109 years ago. This would explain why mitochondria and chloroplasts contain their own DNA, which codes for some of their proteins. Since their initial uptake by a host cell, these organelles have lost much of their own genomes and have become heavily dependent on proteins that are encoded by genes in the nucleus, synthesized in the cytosol, and then imported into the organelle. Conversely, the host cells have become dependent on these organelles for much of the ATP they need to carry out biosyntheses, ion pumping, and movement - as well as requiring selected biosynthetic reactions that occur inside these organelles.
Mitochondria occupy a substantial portion of the cytoplasmic volume of eucaryotic cells, and they have been essential for the evolution of complex animals. Without mitochondria present-day animal cells would be dependent on anaerobic glycolysis for all of their ATP. However, when glucose is converted to pyruvate by glycolysis, only a very small fraction of the total free energy potentially available from the glucose is released. In mitochondria the metabolism of sugars is completed: the pyruvate is imported into the mitochondrion and oxidized by molecular oxygen (O2) to CO2 and H2O. 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 by glycolysis alone.
Rapid changes of shape are observed when a mitochondrion is visualized in a living cell.
(A) Light micrograph of chains of elongated mitochondria in a living mammalian cell in culture. The cell was stained with a vital fluorescent dye (rhodamine 123) that specifically labels mitochondria. (B) Immuno-fluorescence micrograph of the same cell stained (after fixation) with fluorescent antibodies that bind to microtubules. Note that the mitochondria tend to be aligned along microtubules. (Courtesy of Lan Bo Chen.)
During the development of the flagellum of the sperm tail, microtubules wind helically around the axoneme, where they are thought to help localize the mitochondria in the tail; these microtubules then disappear.
) and even fusing
with one another and then separating again. As they move about in the
cytoplasm, they often appear to be associated with microtubules (Figure 14-4), which
may determine the unique orientation and distribution of mitochondria in
different types of cells. Thus the mitochondria in some cells form long moving
filaments or chains, while in others they remain fixed in one position
where they
provide ATP directly to a site of unusually high ATP consumption -
packed between
adjacent myofibrils in a cardiac muscle cell, for example, or wrapped tightly
around the flagellum in a sperm (Figure 14-5).
Although mitochondria are large enough to be seen in the light microscope and were first identified in the nineteenth century, real progress in understanding their function depended on procedures developed in 1948 for isolating intact mitochondria. For technical reasons many biochemical studies have been carried out with mitochondria purified from liver; each liver cell contains 1000 to 2000 mitochondria, which in total occupy roughly a fifth of the cell volume.
These techniques have made it possible to study the different proteins in each mitochondrial compartment. The method shown, which allows the processing of large numbers of mitochondria at the same time, takes advantage of the fact that in media of low osmotic strength water flows into mitochondria and greatly expands the matrix space (yellow). While the cristae of the inner membrane allow it to unfold to accommodate the expansion, the outer membranewhich has no folds to begin withbreaks, releasing a structure composed of only the inner membrane and the matrix.
In the liver an estimated 67% of the total mitochondrial protein is located in the matrix, 21% is located in the inner membrane, 6% in the outer membrane, and 6% in the intermembrane space. As indicated below, each of these four regions contains a special set of proteins that mediate distinct functions. (Courtesy of Daniel S. Friend.)
), the
biochemical composition of each of
the two membranes and of the spaces enclosed by them can be determined. As
described in Figure 14-7, each contains a unique
collection of proteins.
The outer membrane contains many copies of a transport protein called porin (see Chapter 10) , 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, while the intermembrane space is chemically equivalent to the cytosol with respect to the small molecules it contains, the matrix space contains a highly selected set of small molecules.
As we explain in detail later, the major working part of the mitochondrion is the matrix space and the inner membrane that surrounds it. The inner membrane is highly specialized. It contains a high proportion of the "double" phospholipid cardiolipin,which contains four fatty acids and may help make the membrane especially impermeable to ions. It 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 space. 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, in the matrix space. These convolutions greatly increase the area of the inner membrane, so that in a liver cell, for example, it constitutes about a 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 shall largely ignore the differences, however, and focus instead on the enzymes and properties that are common to all mitochondria.
).
);
the acetyl group is then fed
into the citric acid cycle for further degradation, and the process ends
with the
passage of acetyl-derived high-energy electrons along the respiratory chain.
To ensure a continuous supply of fuel for oxidative metabolism, animal cells store fatty acids in the form of fats and glucose in the form of glycogen. Quantitatively, fat is a far more important storage form than glycogen, in part because its oxidation releases more than six times as much energy as the oxidation of an equal mass of glycogen in its hydrated form. An average adult human stores enough glycogen for only about a day of normal activities but enough fat to last for nearly a month. If our main fuel reservoir had to be carried as glycogen instead of fat, body weight would need to be increased by an average of about 60 pounds.
Most of our fat is stored in adipose tissue, from which it is released into the bloodstream for other cells to utilize as needed. The need arises after a period of not eating; even a normal overnight fast results in the mobilization of fat, so that in the morning most of the acetyl CoA entering the citric acid cycle is derived from fatty acids rather than from glucose. After a meal, however, most of the acetyl CoA entering the citric acid cycle comes from glucose derived from food, and any excess glucose is used to replenish depleted glycogen stores or to synthesize fats. (While animal cells readily convert sugars to fats, they cannot convert fatty acids to sugars.)
(A) Electron micrograph of a lipid droplet in the cytoplasm; the droplet contains triacylglycerols, the main form of stored fat. (B) The structure of triacylglycerol, with its glycerol portion in green. (A, courtesy of Daniel S. Friend.)
The droplets are surrounded by mitochondria that oxidize the fatty acids derived from their triacylglycerols.
The cycle is catalyzed by a series of four enzymes in the mitochondrial matrix. Each turn of the cycle shortens the fatty acid chain by two carbons (shown in red), as indicated, and generates one molecule of acetyl CoA and one molecule each of NADH and FADH2. The NADH is freely soluble in the matrix. The FADH2, in contrast, remains tightly bound to the enzyme fatty acyl-CoA dehydrogenase;its two electrons will be rapidly transferred to the respiratory chain in the mitochondrial inner membrane, regenerating FAD.
).
A single very large fat droplet accounts for most of the volume of adipocytes (fat cells), the large cells specialized for fat
storage in adipose tissue. Much
smaller fat droplets are common in cells that rely on the breakdown of
fatty acids for
their energy supply, such as cardiac muscle cells; these droplets are often
closely
associated with mitochondria (Figure 14-10). In
all cells, enzymes in the outer
and inner mitochondrial membranes mediate the movement of fatty acids
derived from fat molecules into the mitochondrial matrix. In the matrix
each fatty
acid molecule (as fatty acyl CoA) is broken down completely by a
cycle of
reactions that trims two carbons at a time from its carboxyl end,
generating one
molecule of acetyl CoA in each turn of the cycle (Figure 14-11). The acetyl CoA
produced is fed into the citric acid cycle to be oxidized further.
Glycogen is the major storage form of carbohydrate in vertebrate cells. It is a polymer of glucose, and each glycogen granule is a single, highly branched molecule. The synthesis and degradation of glycogen are catalyzed by enzymes bound to the granule surface, including the synthetic enzyme glycogen synthase and the degradative enzyme glycogen phosphorylase. (Courtesy of Robert Fletterick and Daniel S. Friend.)
The complex converts pyruvate to acetyl CoA in the mitochondrial matrix; NADH is also produced in this reaction. A, B, and C are the three enzymes pyruvate decarboxylase, lipoamide reductase-transacetylase, and dihydrolipoyl dehydrogenase, whose activities are coupled as shown. The structure of the complex, which is larger than a ribosome, is shown in Figure2-41; the complex also contains a protein kinase and a protein phosphatase that regulate its activity, turning it off whenever ATP levels are high.
); its
synthesis and degradation are
highly regulated according to need. When the need arises, cells break down
glycogen to release glucose 1-phosphate, which is then subjected to glycolysis. The reactions of glycolysis convert the six-carbon
glucose molecule (and related
sugars) to two three-carbon pyruvate molecules, which still retain most of
the energy
that can be derived from the complete oxidation of sugars. This energy is
harvested only after the pyruvate is transported from the cytosol into the
mitochondrial matrix, where it encounters a giant multienzyme complex, the pyruvate dehydrogenase complex. This complex - containing
multiple copies of three enzymes,
five coenzymes, and two regulatory proteins - rapidly converts pyruvate to
acetyl CoA, releasing CO2 as a by-product (Figure 14-13). This acetyl CoA joins the
acetyl CoA produced from fatty acids to fuel the citric acid cycle.In the nineteenth century biologists noticed that in the absence of air (anaerobic conditions) cells produce lactic acid (or ethanol), while in its presence (aerobic conditions) they use O2 to produce CO2 and H2O. Efforts to define the pathways of aerobic metabolism eventually focused on the oxidation of pyruvate and led in 1937 to the discovery of the citric acid cycle, also known as the tricarboxylic acid cycle or the Krebs cycle. The citric acid cycle accounts for about two-thirds of the total oxidation of carbon compounds in most cells, and its end products are CO2 and high-energy electrons, which pass via NADH and FADH2 to the respiratory chain. CO2 is released as a waste product, while the high-energy electrons move along the respiratory chain, eventually combining with O2 to produce H2O.
The intermediates are shown as their free acids, although the carboxyl groups are actually ionized. Each of the indicated steps is catalyzed by a different enzyme located in the mitochondrial matrix. The two carbons from acetyl CoA that enter this turn of the cycle (shadowed in red) will be converted to CO2in subsequent turns of the cycle: it is the two carbons shadowed in blue that are converted to CO2 in this cycle. Three molecules of NADH are formed. The GTP molecule produced can be converted to ATP by the exchange reaction GTP + ADP → GDP + ATP. The molecule of FADH2 formed remains protein-bound as part of the succinate dehydrogenase complex in the mitochondrial inner membrane; this complex feeds the electrons acquired by FADH2 directly to ubiquinone (see below).
). But the
net result, insofar as the acetyl group on acetyl CoA is concerned, is
This reaction also produces one molecule of ATP (via GTP) by the direct transfer of a phosphate from a sugar-phosphate intermediate to GDP; a very similar substrate-level phosphorylation reaction occurs in glycolysis, as explained in Chapter 2.
The most important contribution of the citric acid cycle to metabolism is the extraction of high-energy electrons during the oxidation of the two acetyl carbon atoms to CO2. These electrons, which are transiently held by NADH and FADH2, are quickly passed to the respiratory chain in the mitochondrial inner membrane. FADH2, which is part of the succinate dehydrogenase complex in the inner membrane, passes its electrons directly to the respiratory chain. The NADH, in contrast, forms a soluble pool of reducing equivalents in the mitochondrial matrix and passes on its electrons after a random collision with a membrane-bound dehydrogenase enzyme. We now consider how the energy stored in these electrons is used to synthesize ATP.
In this process of oxidative phosphorylation, the mitochondrial inner membrane serves as a device that converts one form of chemical bond energy to another, changing a major part of the energy of NADH (and FADH2) oxidation into phosphate-bond energy in ATP.
).
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. It was originally believed that the energy for ATP synthesis via the respiratory chain was supplied by the same process that operates during substrate-level phosphorylations: that is, the energy of oxidation was thought to generate a high-energy bond between a phosphate group and some intermediate compound, and the conversion of ADP to ATP was thought to be driven by the energy released when this bond was broken. Despite intensive efforts, however, the expected intermediates could not be detected.
Pyruvate and fatty acids enter the mitochondrion, are broken down to acetyl CoA, and are then metabolized by the citric acid cycle, which produces NADH (and FADH2, which is not shown). In the process of oxidative phosphorylation, high-energy electrons from NADH (and FADH2) are then passed to oxygen by means of the respiratory chain in the inner membrane, producing ATP by a chemiosmotic mechanism.
NADH generated by glycolysis in the cytosol also passes electrons to the respiratory chain (not shown). Since NADH cannot pass across the mitochondrial inner membrane, the electron transfer from cytosolic NADH must be accomplished indirectly by means of one of several "shuttle" systems that transport another reduced compound into the mitochondrion; after being oxidized, this compound is returned to the cytosol, where it is reduced by NADH again.
| The chemiosmotic hypothesis, as proposed in the early 1960s, consisted of four independent postulates. In terms of mitochondrial function they were as follows: | |
| 1. | The mitochondrial respiratory chain in the inner membrane is proton translocating; it pumps H+ out of the matrix space when electrons are transported along the chain. |
| 2. | The mitochondrial ATP synthase also translocates protons across the inner membrane. Being reversible, it can use the energy of ATP hydrolysis to pump H+ across the membrane, but if a large enough electrochemical proton gradient is present, protons flow in the reverse direction through the complex and drive ATP synthesis. |
| 3. | The mitochondrial inner membrane is equipped with a set of carrier proteins that mediate the entry and exit of essential metabolites and selected inorganic ions. |
| 4. | The mitochondrial inner membrane is otherwise impermeable to H+, OH-, and generally to anions and cations. |
. According to the chemiosmotic hypothesis, the high-energy chemical intermediates
are replaced by a link between chemical
processes ("chemi") and transport processes
("osmotic"from the Greek osmos, push)hence chemiosmotic coupling (Table 14-1). As the high-energy electrons
from the hydrogens on NADH and FADH2 are transported down
the respiratory
chain in the mitochondrial inner membrane, the energy released as they pass
from
one carrier molecule to the next is used to pump protons
(H+) across the inner membrane from the mitochondrial matrix
into the intermembrane space. This
creates an electrochemical proton gradient across the
mitochondrial inner
membrane, and the backflow of H+ down this gradient is in
turn used to drive the
membrane-bound enzyme ATP synthase, which catalyzes the
conversion of ADP +
Pi to ATP, completing the process of oxidative phosphorylation.
In the remainder of this section we briefly outline the type of reactions that make oxidative phosphorylation possible, saving the details of the respiratory chain for later.
).
).
The components of two complete hydrogen atoms are lost from the alcohol: a hydride ion is transferred to NAD+, and a proton escapes to the aqueous solution. Only the nicotinamide ring portion of the NAD+ and NADH molecules is shown here (see Figure2-24). The steps illustrated occur on a protein surface, being catalyzed by specific chemical groups on the enzyme alcohol dehydrogenase (not shown). (Modified with permission from P.F. Cook, N.J. Oppenheimer, and W.W. Cleland, Biochemistry20:1817-1825, 1981. © 1981 American Chemical Society.)
. As
this example makes clear, each
molecule of NADH carries a hydride ion (a hydrogen atom plus an
extra
electron, which we can denote as H:-, illustrating each of
its two electrons as a dot),
rather than a single hydrogen atom. Because protons are freely available in
aqueous solutions, however, carrying the hydride ion on NADH is equivalent to
carrying two hydrogen atoms, or a hydrogen molecule
(H:- + H+ →
H2).
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+ + 2 e -). 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 atom to another, each metal atom being tightly bound to a protein molecule, which alters the electron affinity of the metal atom. The various types of electron carriers in the respiratory chain will be discussed in detail later. Most important, the many proteins involved are grouped into three large respiratory enzyme complexes, each containing transmembrane proteins that hold the complex firmly in the mitochondrial inner 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.
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 important, the transfer of electrons is coupled to oriented H+ uptake and release and to allosteric changes in selected protein molecules. The net result is that the energetically favorable flow of electrons pumps H+ across the inner membrane, from the matrix space to the intermembrane space. 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 total proton-motive force across the mitochondrial inner membrane consists of a large force due to the membrane potential (traditionally designated ΔΨ by experts, but designated Δ V in this text) and a smaller force due to the H+ concentration gradient (ΔpH). Both forces act to drive H+ into the matrix space.
).
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 Δ V - 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.
As a high-energy electron is passed along the electron-transport chain, some of the energy released is used to drive three respiratory enzyme complexes that pump H+ out of the matrix space. The resulting electrochemical proton gradient across the inner membrane drives H+ back through the ATP synthase, a transmembrane protein complex that uses the energy of the H+ flow to synthesize ATP from ADP and Pi in the matrix.
).
The charge on each of the transported molecules is indicated for comparison with the membrane potential, which is negative inside, as shown. The outer membrane is freely permeable to all of these compounds. Membrane transport mechanisms are discussed in Chapter 11.
). The electrochemical
H+ gradient is also used to import
Ca2+, which is thought to be important in regulating the
activity of selected
mitochondrial enzymes; the import of
Ca2+ into mitochondria may also be
important for removing Ca2+ from the cytosol when cytosolic
Ca2+ levels become dangerously high.
The more energy from the electrochemical proton gradient is used to transport molecules and ions into the mitochondrion, the less there is to drive the ATP synthase. If isolated mitochondria are incubated in a high concentration of Ca2+, for example, they cease ATP production completely; all the energy in their electrochemical proton gradient is diverted to pumping Ca2+ into the matrix. Similarly, in certain specialized cells the electrochemical proton gradient is short-circuited so that the mitochondria produce heat instead of ATP, as we discuss later. In general, the use of the energy stored in the electrochemical proton gradient is regulated by cells so that it is directed toward those activities that are most needed at the time.
), 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 into and out of a mitochondrion for
recharging
(as ADP) thousands of times a day, keeping the concentration of ATP in a cell
about 10 times higher than that of ADP.
As discussed in Chapter 2, biosynthetic enzymes in cells guide their substrates along specific reaction paths, often driving energetically unfavorable reactions by coupling them to the energetically favorable hydrolysis of ATP (see Figure 2-29). The ATP pool is thereby 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 halted, ATP levels fall and the cell's battery runs down, so that, eventually, energetically unfavorable reactions can no longer be driven by ATP hydrolysis.
It might seem that this state would not be reached until the concentration of ATP is zero, but in fact it is reached much sooner than that, at a concentration of ATP that depends on the concentrations of ADP and Pi. To explain why, we must consider some elementary principles of thermodynamics.
The second law of thermodynamics states that chemical reactions proceed spontaneously in the direction that corresponds to an increase in the disorder of the universe. In Chapter 2 we noted that reactions that release energy to their surroundings as heat (such as the hydrolysis of ATP) tend to increase the disorder of the universe by increasing random molecular motions. For this reason reactions go in the direction that converts free energy (energy that is available to do work) into heat. Thus the reaction A
![[right harpoon over left harpoon]](corehtml/pmc/pmcents/x21CC.gif)
B will go in the direction A →
B when the associated free-energy change,
Δ G, is negative, just as a tensed spring left to
itself will relax and lose its stored energy to its surroundings as heat.
For a
chemical reaction, however, Δ G depends not only on the
energy stored in each
individual molecule but also on the concentrations of the molecules in the
reaction mixture. This is because, for a reversible reaction A
B, a large excess of B over A will tend to drive the reaction in the
direction B
→
A; that is, there will be more molecules making the transition B
→
A than there are making the transition
A →
B. Just how much of a concentration difference is needed to compensate
for a given amount of heat release is not obvious; it depends on entropy changes, which can be calculated as outlined in Panel14-1, pages 668-669.
The Δ G for a given reaction can be written as the sum of two parts: the first, called the standard free-energy change, Δ G°, 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. Δ G° therefore equals the value of Δ G when the molar concentrations of A and B are equal (ln 1 = 0). Chemical equilibrium is reached when the concentration effect is just balanced by the effect of Δ G°, so that there is no net change of free energy to drive the reaction in either direction; then Δ G = 0, and so the concentrations of A and B are such that
which means that there is chemical equilibrium when
).
It is Δ G, not Δ G°, that indicates how far a reaction is from equilibrium and determines if 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 Pi, the ATP-hydrolysis reaction in cells is kept very far from equilibrium and Δ G is correspondingly very negative. Without this disequilibrium ATP hydrolysis could not be used to direct the reactions of the cell, and many biosynthetic reactions would run backward rather than forward.
By means of oxidative phosphorylation, each pair of electrons in NADH is thought to provide energy for the formation of about 2.5 molecules of ATP. The pair of electrons in FADH2, being at a lower energy, generates only about 1.5 ATP molecules. In all, about 10 molecules of ATP can be formed from each molecule of acetyl CoA that enters the citric acid cycle, which means that about 20 ATP molecules are produced from 1 molecule of glucose and 84 ATP molecules from 1 molecule of palmitate, a 16-carbon fatty acid. If one includes the energy-yielding reactions that occur before acetyl CoA is formed, the complete oxidation of 1 molecule of glucose gives a net yield of about 30 ATPs, while the complete oxidation of 1 molecule of palmitate gives a net yield of about 110 ATPs. These numbers are approximate maximal values. As previously discussed, the actual amount of ATP made in the mitochondrion depends on what fraction of the electrochemical gradient energy is used for purposes other than ATP synthesis.
When the free-energy changes for burning fats and carbohydrates directly into CO2 and H2O are compared to the total amount of energy generated and stored in the phosphate bonds of ATP during the corresponding biological oxidations, it is seen that the efficiency with which oxidation energy is converted into ATP bond energy is often greater than 40%. This is considerably better than the efficiency of most nonbiological energy-conversion devices. If cells worked with the efficiency of an electric motor or a gasoline engine (10-20%), an organism would have to eat voraciously in order to maintain itself. Moreover, since wasted energy is liberated as heat, large organisms would need more efficient mechanisms for giving up heat to the environment.
Students sometimes wonder why the chemical interconversions in cells follow such complex pathways. The oxidation of sugars to CO2 plus H2O could certainly be accomplished more directly, eliminating the citric acid cycle and many of the steps in the respiratory chain. Although this would have made respiration easier to learn, it would have been a disaster for the cell. Oxidation produces huge amounts of free energy, which can be utilized efficiently only in small bits. The complex oxidative pathways involve many intermediates, each differing only slightly from its predecessor. The energy released is thereby parceled out into small packets that can be efficiently converted to high-energy bonds in useful molecules such as ATP and NADH by means of coupled reactions (see Figure2-17).
The mitochondrion carries out most cellular oxidations and produces the bulk of the animal cell's ATP. The mitochondrial matrix space 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 CO2 through the citric acid cycle. Large amounts of NADH (and FADH2) are produced by these oxidation reactions. The energy available from combining oxygen with the reactive electrons carried by NADH and FADH2 is harnessed by an electron-transport chain in the mitochondrial inner 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 transmembrane gradient in turn is used both to synthesize ATP and to drive the active transport of selected metabolites across the mitochondrial inner membrane. The combination of these reactions is responsible for an efficient ATP-ADP exchange between the mitochondrion and the cytosol that keeps the cell's ATP pool highly charged, so that ATP can be used to drive many of the cell's energy-requiring reactions.
Having considered in general terms how mitochondria function, let us now look in more detail at the respiratory chain - the electron-transport chain that is so crucial to all oxidative metabolism. Most of the elements of the chain are intrinsic components of the inner mitochondrial membrane, and they provide some of the clearest examples of the many complicated interactions that can occur among the individual proteins located in a biological membrane.
The particles are pieces of broken-off cristae that form closed vesicles.
This preparation has been negatively stained. (Courtesy of Efraim Racker.)
). When these
submitochondrial particles are examined in
an electron microscope, their outside surfaces are seen to be studded with
tiny spheres attached to the membrane by stalks (Figure 14-24). In intact
mitochondria these lollipoplike structures are located on the inner (matrix) side of the inner membrane. Thus the
submitochondrial particles are inside-out vesicles of
inner membrane, with what was previously their matrix-facing surface
exposed to
the surrounding medium. As a result, they can readily be provided with the
membrane-impermeable metabolites that would normally be present in the
matrix space. When NADH, ADP, and inorganic phosphate are added, such
preparations transport electrons from NADH to
O2 and couple this oxidation to ATP
synthesis, catalyzing the reaction ADP + Pi→
ATP. This cell-free system provides
an assay that makes it possible to purify the many proteins responsible for
oxidative phosphorylation in a functional form.The first experiments to show that the various membrane proteins that catalyze oxidative phosphorylation can be separated without destroying their activity were performed in 1960. The tiny protein spheres studding the surface of submitochondrial particles were stripped from the particles, leaving the stem of the lollipop and the other inner membrane proteins still in the particle membrane. The stripped particles could still oxidize NADH in the presence of oxygen, but they could no longer synthesize ATP. On the other hand, the purified spheres on their own acted as ATPases, hydrolyzing ATP to ADP and Pi. When the purified spheres (referred to as F1ATPase) were added back to stripped submitochondrial particles, however, the reconstituted particles once again made ATP from ADP and Pi.
As indicated, the F1ATPase portion is formed from multiple subunits (Greek letters), as is the transmembrane H+ carrier.
), which is now
known as
ATP synthase (also called F0F1ATPase). ATP synthase
constitutes about 15% of the total inner
membrane protein, and very similar enzyme complexes are present in both
chloroplast and bacterial membranes. The transmembrane portion of the
protein complex
acts as a H+ carrier, and the
F1ATPase portion (the lollipop head) normally
synthesizes ATP when protons pass through it down their electrochemical
gradient.
When separated from the H+ carrier, however, the
F1ATPase goes into reverse and catalyzes only ATP hydrolysis.
By combining a light-driven bacterial proton pump (bacteriorhodopsin), an ATP synthase purified from ox heart mitochondria, and phospholipids, vesicles were produced that synthesized ATP in response to light.
).
Be-cause a direct interaction between a bacterial
H+ pump and a mammalian ATP synthase seems highly unlikely,
this experiment strongly suggests that in
mitochondria the proton translocation driven by electron transport and the
ATP synthesis are separate events.
The ATP synthase can either synthesize ATP by harnessing the proton-motive force (top) or pump protons against their electrochemical gradient by hydrolyzing ATP (bottom). As explained in the text, the direction of operation at any given instant depends on the net free-energy change for the coupled processes of H+translocation across the membrane and the synthesis of ATP from ADP and Pi.
); the
Δ Gfor ATP synthesis is the negative of this value. The
Δ G for proton translocation across the membrane
is proportional to the proton-motive force. The conversion factor
between them is the faraday. Thus,
Δ G
H+ = -0.023 (proton-motive
force), where Δ G
H+ is in kilocalories per
mole (kcal/mole) and the proton-motive force is in millivolts (mV). For
an electrochemical H+ gradient of 200 mV,
Δ G
H+ = -4.6 kcal/mole.
). It thus acts as a reversible coupling device,interconverting
electrochemical-proton-gradient and
chemical-bond energies. Its direction of action depends on the balance between
the steepness of the electrochemical proton gradient and the local
Δ G for ATP
hydrolysis.
) to make most of the
cell's ATP. The exact number of
protons needed to make each ATP molecule is not known with certainty. To
facilitate
the calculations to be described below, however, we shall assume that one
molecule of ATP is made by the ATP synthase for every three protons driven
through it.
). The value of
Δ G
3H+, on the other hand, is proportional to
the value of the proton-motive force across the inner mitochondrial membrane.
The following example will help to explain how the balance between these two
free-energy changes affects the ATP synthase.
, a single
H+ moving into the matrix down an electrochemical gradient
of 200 mV liberates 4.6 kcal/mole of free
energy, while the movement of three protons liberates three times this much
free energy (Δ G
3H+ = -13.8 kcal/mole). Thus, if
the proton-motive force remains
constant at 200 mV, the ATP synthase will synthesize ATP until a ratio of ATP
to ADP and Pi is reached where
Δ G
ATP synthesis is just equal to +13.8
kcal/mole
(here Δ G
ATP synthesis +
Δ G
3H+ = 0). At this point there will be no
further net ATP
synthesis or hydrolysis by the ATP synthase.
), and ATP synthase
will begin to synthesize ATP again to restore the original ATP:ADP ratio.
Alternatively, if the proton-motive force drops suddenly and is then
maintained at a
constant 160 mV, Δ G
3H+ will change to
-11.0 kcal/mole. As a result, ATP synthase will
start hydrolyzing some of the ATP in the matrix until a new balance of ATP
to ADP
and Pi is reached (where Δ G
ATP
synthesis = +11.0 kcal/mole) and so on.
In many bacteria ATP synthase is routinely reversed in a transition between aerobic and anaerobic metabolism, as we shall see later. The reversibility of the ATP synthase is a property shared by other membrane proteins that couple ion movement to ATP synthesis or hydrolysis. Both the Na+-K+ pump and the Ca2+ pump described in Chapter 11, for example, hydrolyze ATP and use the energy released to pump specific ions across a membrane. If either of these pumps is exposed to an abnormally steep gradient of the ions it transports, however, it will act in reverse - synthesizing ATP from ADP and Pi instead of hydrolyzing it. Thus, like ATP synthase, such pumps are able to convert the electrochemical energy stored in a transmembrane ion gradient directly into phosphate bond energy in ATP.
The respiratory chain embedded in the inner mitochondrial membrane normally generates the electrochemical proton gradient that drives ATP synthesis. The ability of the respiratory chain to translocate H+ outward from the matrix space can be demonstrated experimentally under special conditions. A suspension of isolated mitochondria, for example, can be provided with a suitable substrate for oxidation, and the H+ flow through ATP synthase can be blocked. In the absence of air the injection of a small amount of oxygen into such a preparation causes a brief burst of respiration, which lasts for 1 to 2 seconds before all the oxygen is consumed. During this respiratory burst a sudden acidification of the medium resulting from the extrusion of H+ from the matrix space can be measured with a sensitive pH electrode.
In a similar experiment carried out with a suspension of submitochondrial particles, the medium becomes more basic when oxygen is injected, since H+ is pumped into each vesicle because of its inside-out orientation.
Many of the electron carriers in the respiratory chain absorb visible light and change color when they are oxidized or reduced. In general, each has an absorption spectrum and reactivity that is distinct enough to allow its behavior to be traced spectroscopically even in crude mixtures. It was therefore possible to purify these components long before their exact functions were known. Thus the cytochromes were discovered in 1925 as compounds that undergo rapid oxidation and reduction in living organisms as disparate as bacteria, yeasts, and insects. By observing cells and tissues with a spectroscope, three types of cytochromes were identified by their distinctive absorption spectra and designated cytochromes a, b, and c. This nomenclature has survived even though cells are now known to contain several cytochromes of each type and the classification into types is not functionally important.
The porphyrin ring is shown in blue. There are five different cytochromes in the respiratory chain. Because the hemes in different cytochromes have slightly different structures and are held by their respective proteins in different ways, each of the cytochromes has a different affinity for an electron.
). The iron atom (orange) on the
bound heme
(blue) can carry a single electron (see also Figure3-59).
). A related porphyrin
ring is responsible for the red color of blood and the green color of leaves,
being bound to iron in hemoglobin and to magnesium in chlorophyll. The best
understood of the many proteins in the respiratory chain is cytochrome c, whose three-dimensional structure has been
determined by x-ray crystallography (Figure 14-29).
(A) A center of the 2Fe2S type. (B) A center of the 4Fe4S type. Although they contain multiple iron atoms, each iron-sulfur center can carry only one electron at a time. There are more than six different iron-sulfur centers in the respiratory chain.
).
There are more iron-sulfur centers than cytochromes in the
respiratory chain, but their spectroscopic detection requires electron spin
resonance (ESR) spectroscopy, and they are less well characterized.
Each of these electron carriers in the respiratory chain picks up one H+ from the aqueous environment for every electron it accepts, and it can carry either one or two electrons as part of a hydrogen atom (yellow). When it donates its electrons to the next carrier in the chain, these protons are released. In mitochondria the quinone is ubiquinone (coenzyme Q), shown here; the long hydrophobic tail, which confines ubiquinone to the membrane, consists of 6 to 10 five-carbon isoprene units, depending on the organism. The corresponding electron carrier in plants is plastoquinone, which is almost identical. For simplicity, both ubiquinone and plastoquinone will normally be referred to as quinone and abbreviated as Q.
).
The extent of oxidation of electron carriers a, b, c, and d is continuously monitored by following their distinct spectra, which differ in their oxidized and reduced states. In this schematic an increased degree of oxidation is indicated by a darker red. (A) Under normal conditions, where oxygen is abundant, all carriers are in a partially oxidized state. Addition of a specific inhibitor causes the downstream carriers to become more oxidized (red) and the upstream carriers to become more reduced. (B) In the absence of oxygen all carriers are in their fully reduced state (gray). The sudden addition of oxygen converts each carrier to its partially oxidized form with a delay that is greatest for the most upstream carriers.
), 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 size and shape of each complex is shown, as determined from images of two-dimensional crystals (crystalline sheets) viewed in the electron microscope at various tilt angles. During the transfer of two electrons from NADH to oxygen (red lines) ubiquinone and cytochrome c serve as carriers between the complexes.
). As we shall see, each of
these complexes acts as an electron-transport-driven
H+ pump; they were initially characterized, however, in
terms of the electron carriers that they interact
with and contain.
The NADH dehydrogenase complex is the largest of the respiratory enzyme complexes, with a mass of about 800,000 daltons and more than 22 polypeptide chains. It accepts electrons from NADH and passes them through a flavin and at least five iron-sulfur centers to ubiquinone, which transfers its electrons to a second respiratory enzyme complex, the b-c1 complex.
The cytochrome b-c1 complex contains at least 8 different polypeptide chains and is thought to function as a dimer of about 500,000 daltons. Each monomer contains three hemes bound to cytochromes and an iron-sulfur protein. The complex accepts electrons from ubiquinone and passes them on to cytochrome c, which carries its electron to the cytochrome oxidase complex.
The cytochrome oxidase complex (cytochrome aa3) is the best characterized of the three complexes. It is isolated as a dimer of about 300,000 daltons; each monomer contains at least 9 different polypeptide chains, including two cytochromes and two copper atoms. The complex accepts electrons from cytochrome c and passes them to oxygen.
The cytochromes, iron-sulfur centers, and copper atoms can carry only one electron at a time. Yet each NADH donates two electrons, and each O2 molecule must receive four electrons to produce water. There are several electron-collecting and electron-dispersing points along the electron-transport chain where these changes in electron number are accommodated.
(A) The arrangement of electron carriers in cytochrome oxidase. Subunit I, which has 12 membrane-spanning alpha helices, contains two heme-linked iron atoms; one of these serves as an electron queuing point that feeds electrons into the bimetallic center (boxed), which is formed by the other iron and a closely opposed copper atom. Note that four protons are pumped out of the matrix for each O2 molecule that reacts and that this requires a total of four electrons. (B) An enlarged view of the bimetallic iron-copper center with O2 bound. (C) An outline of the pathway used for oxygen reduction at the bimetallic center, giving some idea of the complexity of the reactions involved. Electrons are shown as red dots until they become incorporated into hydrogen atoms (yellow). (Based on G.T. Babcock and M. Wikström, Nature 356:301-309, 1992. © 1992 Macmillan Magazines Ltd.)
), where it remains clamped between a
heme-linked iron atom and a
copper atom until it has picked up a total of four electrons; only then can
the two
oxygen atoms of the oxygen molecule be safely released as two molecules of
water (Figure 14-34C).
).
The cytochrome oxidase reaction is estimated to account for 90% of the total oxygen uptake in most cells. Cyanide and azide are toxic to cells because they bind tightly to this complex and thereby block all electron transport.
The two components that carry electrons between the three major enzyme complexes of the respiratory chain - ubiquinone and cytochrome c - diffuse rapidly in the plane of the inner mitochondrial membrane. The expected rate of random collisions between these mobile carriers and the enzyme complexes can account for the observed rates of electron transfer (each complex donates and receives an electron about once every 5 to 20 milliseconds). Thus there is no need to postulate a structurally ordered chain of electron-transfer proteins in the lipid bilayer; indeed, the three enzyme complexes appear to exist as independent entities in the plane of the inner membrane, and the ordered transfer of electrons is due entirely to the specificity of the functional interactions among the components of the chain.
, and there is a net flow of
electrons from NADH dehydrogenase to cytochrome oxidase because each of the
enzyme complexes in the sequence has a higher affinity for electrons than its
predecessor. The affinity of a molecule for electrons is its redox potential. The changes in redox potential from one
electron carrier to the next are exploited to pump
proteins out of the mitochondrial matrix, as we now discuss.Pairs of compounds such as H2O and 1/2 O2, or NADH and NAD+, are called conjugate redox pairs, since one compound is converted to the other by adding one or more electrons plus one or more protons - the protons being readily available in any aqueous solution. Thus, for example,
Many readers will know that a 50-50 (equimolar) mixture of the members of a conjugate acid-base pair acts as a buffer, maintaining a defined "H+ pressure," or pH, which is a measure of the dissociation constant of the acid. In exactly the same way a 50-50 mixture of the members of a conjugate redox pair maintains a defined "electron pressure," or redox (reduction-oxidation) potential, E, that is a measure of the electron carrier's affinity for electrons.
By placing electrodes in contact with solutions that contain the appropriate conjugate redox pairs, one can measure the redox potential of each of the various electron carriers that participate in biological oxidation-reduction reactions. For biological systems each redox potential is determined at pH 7.0, where [H+] = 10-7M. Those pairs of compounds that have the most negative redox potentials have the weakest affinity for electrons and therefore contain carriers with the strongest tendency to donate electrons, whereas pairs that have the most positive redox potentials have the strongest affinity for electrons and contain carriers with the strongest tendency to accept electrons. Thus a 50-50 mixture of NADH and NAD+ has a redox potential of -320 mV, indicating that NADH has a strong tendency to donate electrons; a 50-50 mixture of H2O and 1/2 O2 has a redox potential of +820 mV, indicating that O2 has a strong tendency to accept electrons.
Redox potentials can be readily determined for all the electron carriers in the respiratory chain that can be distinguished by their spectra, and they can be shown to increase as one passes along the chain of electron carriers. As most cytochromes have higher redox potentials than iron-sulfur centers, they generally serve as electron carriers near the O2 end of the respiratory chain, whereas the iron-sulfur proteins serve as carriers near the NADH end.
The standard free-energy change, Δ G°(in kilocalories per mole), for the transfer of the two electrons donated by an NADH molecule can be obtained from the right-hand ordinate (Δ G° = - n(0.023) Δ E'0, where n is the number of electrons transferred across a redox potential change of Δ E'0 mV). Electrons flow through an enzyme complex by passing in sequence to the four or more electron carriers in each complex. As indicated, part of the favorable free-energy change is harnessed by each enzyme complex to pump H+ across the mitochondrial inner membrane. Although the number of H+pumped per electron (n) is uncertain, it is estimated that the NADH dehydrogenase and b-c1 complexes each pump two H+ per electron, whereas the cytochrome oxidase complex pumps one.
) and
by the citric acid cycle (see Figure 14-14), are
passed directly to
ubiquinone, and they therefore cause less
H+ pumping than the two electrons transported from NADH (not
shown).
. The potentials drop in
three large steps, one across
each major enzyme complex. The change in redox potential between any two
electron carriers is directly proportional to the free energy
released by an electron
transfer between them (see Figure 14-35). Each
complex acts as an
energy-conversion device, harnessing this free-energy change to pump
H+ across the inner membrane, thereby creating an
electrochemical proton gradient as electrons
pass through. This conversion can be demonstrated by incorporating each
purified complex separately into liposomes: when an appropriate electron donor
and acceptor is added so that electrons can pass through the complex,
H+ is translocated across the liposome membrane.
). Since ubiquinone
is freely mobile in the lipid
bilayer, it could accept electrons near the inside surface of the membrane and
donate them to the bc1 complex near the outside surface,
thereby transferring one
H+ across the bilayer for every electron transported. Two
protons are pumped
per electron in the bc1 complex, however, and there is
evidence for a so-called Q-cycle, in which ubiquinone is recycled through the complex in
an ordered
way that makes this two-for-one transfer possible.
This general model for energy-driven H+ pumping is based on the mechanism that is thought to be utilized by bacteriorhodopsin. The transmembrane protein shown is driven through a cycle of three conformations, denoted here as A, B, and C. In conformation C the protein has a low affinity for H+, causing it to release an H+ on the outside of the lipid bilayer; in conformation A the protein has a high affinity for H+, causing it to pick up an H+ on the inside of the lipid bilayer. As indicated, the transition from conformation B to conformation C is energetically unfavorable but is driven by being coupled to an energetically favorable reaction occurring elsewhere on the protein (blue arrow). The other conformational changes lead to states of lower energy and proceed spontaneously. The cycle A → B → C → A therefore goes only one way, causing H+ to be pumped from the inside to the outside. For bacteriorhodopsin the energy for the transition B → C is provided by light, whereas in the mitochondria this energy is provided by electron transport.
.Since the 1940s several substances, such as 2,4-dinitrophenol, have been known to act as uncoupling agents, uncoupling electron transport from ATP synthesis. The addition of these low-molecular-weight organic compounds to cells stops ATP synthesis by mitochondria without blocking their uptake of oxygen. In the presence of an uncoupling agent electron transport and H+ pumping continue at a rapid rate, but no H+ gradient is generated. The explanation for this effect is both simple and elegant: uncoupling agents are lipid-soluble weak acids that act as H+ carriers (H+ ionophores) and provide a pathway in addition to the ATP synthase for the flow of H+ across the inner mitochondrial membrane. As a result of this "short-circuiting," the proton-motive force is dissipated completely, and ATP can no longer be made.
).
Respiratory control is just one part of an elaborate interlocking system of feedback controls that coordinates the rates of glycolysis, fatty acid breakdown, the citric acid cycle, and electron transport. The rates of all of these processes are adjusted to the ATP:ADP ratio, increasing whenever increased utilization of ATP causes the ratio to fall. The ATP synthase in the inner mitochondrial membrane, for example, works faster as the concentrations of its substrates ADP and Pi 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.
, for example), act to adjust
the rates of NADH production
to the rate of NADH utilization by the respiratory chain and so on. As a
result
of these many control mechanisms, the body oxidizes fats and sugars 5 to 10
times more rapidly during a period of strenuous exercise than during a
period of rest.In some specialized fat cells mitochondrial respiration is normally uncoupled from ATP synthesis. In these cells, known as brown fat cells, most of the energy of oxidation is dissipated as heat rather than being converted into ATP. The inner membranes of the large mitochondria in these cells contain a special transport protein that allows protons to move down their electrochemical gradient without activating ATP synthase. As a result, the cells oxidize their fat stores at a rapid rate and produce more heat than ATP. Tissues containing brown fat thereby serve as "heating pads" that revive hibernating animals and protect sensitive areas of newborn human babies from the cold.
Bacteria use enormously diverse energy sources. Some, like animal cells, are aerobic and synthesize ATP from sugars that they oxidize to CO2 and H2O by glycolysis and the citric acid cycle through a respiratory chain in their plasma membrane similar to that in the mitochondrial inner membrane. Others are strict anaerobes, deriving their energy either from glycolysis alone (by fermentation) or, in addition, from an electron-transport chain that employs a molecule other than oxygen as the final electron acceptor. The alternative electron acceptor can be a nitrogen compound (nitrate or nitrite), a sulfur compound (sulfate or sulfite), or a carbon compound (fumarate or carbonate), for example. The electrons are transferred to these acceptors by a series of electron carriers in the plasma membrane that are comparable to those in mitochondrial respiratory chains.
Despite this diversity, the plasma membrane of the vast majority of bacteria contains an ATP synthase that is very similar to that in mitochondria (and chloroplasts). In aerobic bacteria the electron-transport chain pumps H+ out of the cell and thereby establishes a proton-motive force that drives the ATP synthase to make ATP. In anaerobic bacteria that lack an electron-transport chain, the ATP synthase works in reverse, using the ATP produced by glycolysis to pump H+ and establish a proton-motive force across the bacterial plasma membrane.
A proton-motive force generated across the plasma membrane pumps nutrients into the cell and expels sodium. In (A) the electrochemical proton gradient is generated in an aerobic bacterium by a respiratory chain and is then used by ATP synthase to make ATP and to transport some nutrients into the cell. In (B) the same bacterium growing under anaerobic conditions can derive its ATP from glycolysis. Part of this ATP is hydrolyzed by ATP synthase to establish the transmembrane proton-motive force that drives transport processes.
). In
animal cells, by contrast, most inward transport across the plasma membrane
is driven by the Na+ gradient established by the
Na+-K+ ATPase.
Some unusual bacteria have adapted to live in a very alkaline environment and yet must maintain their cytoplasm at a physiological pH. For these cells any attempt to generate an electrochemical H+ gradient would be opposed by a large H+ concentration gradient in the wrong direction (H+ higher inside than outside). Presumably for this reason, at least some of these bacteria substitute Na+ for H+ in all of their chemiosmotic mechanisms. The respiratory chain pumps Na+ out of the cell, the transport systems and flagellar motor are driven by an inward flux of Na+, and a Na+-driven ATP synthase synthesizes ATP. The existence of such bacteria demonstrates that the principle of chemiosmosis is more fundamental than the proton-motive force on which it is normally based.
The respiratory chain in the inner mitochondrial membrane contains three major enzyme complexes through which electrons pass on their way from NADH to O2. Each of these can be purified, inserted into synthetic lipid vesicles, and then shown to pump H+ when electrons are transported through it. In the native membrane the mobile electron carriers ubiquinone and cytochrome c complete the electron-transport chain by shuttling between the enzyme complexes. The path of electron flow is NADH → NADH dehydrogenase complex → ubiquinone → b-c1 complex → cytochrome c → cytochrome oxidase complex → molecular oxygen (O2).
The respiratory enzyme complexes couple the energetically favorable transport of electrons to the pumping of H+ out of the matrix. The resulting electrochemical proton gradient is harnessed to make ATP by another transmembrane protein complex, ATP synthase, through which H+ flows back into the matrix. The ATP synthase is a reversible coupling device that normally converts a backflow of H+ into ATP phosphate-bond energy by catalyzing the reaction ADP + Pi → ATP, but it can also work in the opposite direction and hydrolyze ATP to pump H+ if the electrochemical proton gradient is reduced. Its universal presence in mitochondria, chloroplasts, and bacteria testifies to the central importance of chemiosmotic mechanisms in cells.
All animals and most microorganisms rely on the continual uptake of large amounts of organic compounds from their environment. These compounds provide both the carbon skeletons for biosynthesis and the metabolic energy that drives all cellular processes. It is believed that the first organisms on primitive earth had access to an abundance of organic compounds produced by geochemical processes (see Chapter 1) but that most of these original compounds were used up billions of years ago. Since that time virtually all of the organic materials required by living cells have been produced by photosynthetic organisms, including many types of photosynthetic bacteria. The most advanced photosynthetic bacteria are the cyanobacteria, which have minimal nutrient requirements. They use electrons from water and the energy of sunlight to convert atmospheric CO2 into organic compounds. In the course of splitting water [in the reaction nH2O + nCO2 -light→ (CH2O) n + nO2], they liberate into the atmosphere the oxygen required for oxidative phosphorylation. As we shall see, it is thought that the evolution of cyanobacteria from more primitive photosynthetic bacteria first made possible the development of aerobic life forms.
In plants, which developed later, photosynthesis occurs in a specialized intracellular organelle - the chloroplast. Chloroplasts carry out photosynthesis during the daylight hours. The products of photosynthesis are used directly by the photosynthetic cells for biosynthesis and are also converted to a low-molecular-weight sugar (usually sucrose) that is exported to meet the metabolic needs of the many nonphotosynthetic cells of the plant. Alternatively, the products can be stored as an osmotically inert polysaccharide (usually starch) that is kept available as a source of sugar for future use.
Biochemical evidence suggests that chloroplasts are descendants of oxygen-producing photosynthetic bacteria that were endocytosed and lived in symbiosis with primitive eucaryotic cells. Mitochondria are also generally believed to be descended from endocytosed bacteria. The many differences between chloroplasts and mitochondria are thought to reflect their different bacterial ancestors as well as their subsequent evolutionary divergence. Nevertheless, the fundamental mechanisms involved in light-driven ATP synthesis in chloroplasts and in respiration-driven ATP synthesis in mitochondria are very similar.
| Organism | Tissue or Cell Type | DNA Molecules per Organelle | Organelles per Cell | Organelle DNA as Percent of Total Cellular DNA |
|---|---|---|---|---|
| Mitochondrial DNA | ||||
| Rat | liver | 5-10 | 1000 | 1 |
| Yeast* | vegetative | 2-50 | 1-50 | 15 |
| Frog | egg | 5-10 | 107 | 99 |
| Chloroplast DNA | ||||
| Chlamydomonas | vegetative | 80 | 1 | 7 |
| Maize | leaves | 20-40 | 20-40 | 15 |
The large variation in the number and size of mitochondria per cell in yeast is due to mitochondrial fusion and fragmentation.
(A) A proplastid from a root tip cell of a bean plant. Note the double membrane; the inner membrane gives rise to the relatively sparse internal membranes. (B) Three amyloplasts (a form of leucoplast), or starch-storing plastids, in a root tip cell of soybean. (From B. Gunning and M. Steer, Ultrastructure and the Biology of Plant Cells. London: Arnold, 1975.)
).
Proplastids develop according to the requirements of each differentiated
cell, and which
type 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 that contain 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.
), which
accumulates starch in storage tissues. 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 exploited their plastids in the cellular compartmentalization of intermediary metabolism. Plastids produce more than the energy and reducing power (as ATP and NADPH) that is used for the plant's biosynthetic reactions. Purine and pyrimidine, most amino acid, 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.
), but these are probably joined
into a single, highly
folded membrane in each chloroplast. As indicated, the individual
thylakoids are interconnected, and they tend to stack to form aggregates
called grana.
(A) A wheat leaf cell in which a thin rim of cytoplasm containing chloroplasts surrounds a large vacuole. (B) A thin section of a single chloroplast, showing the starch granules and lipid droplets that have accumulated in the stroma as a result of the biosyntheses occurring there. (C) A high-magnification view of a granum, showing its stacked thylakoid membrane. (Courtesy of K. Plaskitt.)
and 14-40). They have a highly
permeable outer membrane, a much less permeable inner membrane, in which
special
carrier proteins are embedded, and a narrow intermembrane space. The
inner membrane surrounds a large space called the
stroma, which is analogous to the mitochondrial matrix and contains various
enzymes, ribosomes, RNA, and DNA.
). The lumen of each
thylakoid is thought to be connected with the lumen ofother
thylakoids, thereby defining a third internal compartment called the thylakoid space, which is separated from the stroma by the thylakoid membrane.
The chloroplast is generally much larger and contains a thylakoid membrane and thylakoid space. The mitochondrial inner membrane is folded into cristae.
.
Superficially, the chloroplast
resembles a greatly enlarged mitochondrion in which the cristae have been
converted
into a series of interconnected submitochondrial particles in the matrix
space.
The knobbed end of the chloroplast ATP synthase, where ATP is made, protrudes
from the thylakoid membrane into the stroma, just as it protrudes into the
matrix
from the membrane of each mitochondrial crista.The many reactions that occur during photosynthesis 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 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, with the liberation of O2. During the electron-transport process H+ is pumped across the thylakoid membrane, and the resulting proton-motive force 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 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 in the leaves of the plant; from there it is exported to other tissues as a source of both organic molecules and energy for growth.
Water is oxidized and oxygen is released in the photosynthetic electron-transfer reactions, while carbon dioxide is assimilated (fixed) to produce carbohydrate in the carbon-fixation reactions.
). We shall see, however,
that elaborate feedback mechanisms interconnect the two in order to balance
biosynthesis. Changes in the cell's ATP and NADPH requirements, for example,
regulate the production of these two molecules in the thylakoid membrane, and
several of the chloroplast enzymes required for carbon fixation
are inactivated in
the dark and reactivated by light-stimulated electron-transport processes.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 and must be coupled to other, very favorable reactions to drive it.
This reaction, in which carbon dioxide is converted into organic carbon, is catalyzed in the chloroplast stroma by the abundant enzyme ribulose bisphosphate carboxylase. The product, 3-phosphoglycerate, is also an important intermediate in glycolysis: the two carbon atoms shaded in blue are used to produce phosphoglycolate when the enzyme adds oxygen instead of CO2 (see below).
:
CO2 from the atmosphere combines with the five-carbon
compound ribulose
1,5-bisphosphate plus water to give 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 (~500,000 daltons). Since each
copy 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 copies are needed.
Ribulose bisphosphate carboxylase often constitutes more than 50% of the total
chloroplast protein and is thought to be the most abundant protein on earth.
The number of carbon atoms in each type of molecule is indicated in the white box. There are many intermediates between glyceraldehyde 3-phosphate and ribulose 5-phosphate, but they have been omitted here for clarity. The entry of water into the cycle is also not shown.
). But to produce a supply of
ribulose 1,5-bisphosphate requires a series of reactions that use up large
amounts
of NADPH and ATP. The elaborate pathway by which this compound is
regenerated was worked out in one of the most successful early applications of
radioisotopes. As outlined in Figure 14-44, 3
molecules of
CO2 are fixed by ribulose bisphosphate carboxylase to
produce 6 molecules of 3-phosphoglycerate
(containing 6 x 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 x 5 = 15 carbon atoms). This
leaves 1
molecule of glyceraldehyde 3-phosphate (3 carbon atoms) as the
net gain. In this
carbon-fixation cycle (or Calvin-Benson cycle), 3 molecules of ATP and 2
molecules
of NADPH are consumed for each CO2 molecule converted into
carbohydrate. The net equation is
3CO2 + 9ATP + 6NADPH + water → glyceraldehyde 3-phosphate + 8Pi + 9ADP + 6NADP+
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 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 reversal of several reactions in glycolysis (see Figure 2-21). Glucose 1-phosphate is then converted to the sugar nucleotide UDP-glucose, and this combines with 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.
) 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.Although ribulose bisphosphate carboxylase preferentially adds CO2 to ribulose 1,5-bisphosphate, it can use O2 in addition to CO2, and if the concentration of CO2 is low, it will add O2 instead. 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.
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 "pump"
CO2 into the bundle-sheath cells, supplying the ribulose
bisphosphate
carboxylase with a high concentration of
CO2, which greatly reduces photorespiration.
The cells with green cytosol in the leaf interior contain chloroplasts that carry out the normal carbon-fixation cycle. In C4 plants the mesophyll cells are specialized for CO2 pumping rather than for carbon fixation, and they thereby create a high CO2:O2 ratio in the bundle-sheath cells, which are the only cells in these plants where the carbon-fixation cycle occurs. The vascular bundles carry the sucrose made in the leaf to other tissues.
)
because they
capture CO2 directly into the three-carbon compound
3-phosphoglycerate.
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 may be much less than the energy lost by photorespiration in C3 plants, and so C4 plants have a potential advantage. Moreover, because C4 plants can carry out photosynthesis at a lower concentration of CO2 inside the leaf, they need to open their stomata less and therefore can fix about twice as much net carbon as C3 plants per unit of water lost.
). Electrons are delocalized over
the bonds shown
in color.
The light energy absorbed by an isolated chlorophyll molecule is completely released as light and heat by process 1. In photosynthesis, by contrast, chlorophylls undergo process 2 in the antenna complex and process 3 in the reaction center, as described in the text.
). The
required energy is derived from
sunlight absorbed by chlorophyll molecules (Figure 14-46). 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. Such an excited molecule is unstable and will tend 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; or (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,
Figure 14-47). The last two mechanisms are exploited in
the process of photosynthesis.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: a photochemical reaction center consisting of a complex of proteins and chlorophyll molecules that enable light energy to be converted into chemical energy and an antenna complex consisting of pigment molecules that capture light energy and feed it to the reaction center.
Molecules A (electron donor) and B (electron acceptor) differ according to the photosystem.
).
), and
the excited electron is then transferred stepwise from the special pair to
the quinone (see Figure 14-50
).
). By moving the
high-energy electron rapidly
away from the chlorophylls, the reaction center transfers it to an environment
where it is much more stable. The electron is thereby suitably positioned for
subsequent photochemical reactions, which require more time to complete.
). This structure, combined
with kinetic data, provides the best picture we have of the initial
electron-transfer reactions that underlie photosynthesis.
, plus an
exchangeable quinone (QB) and a freely mobile quinone (Q)
dissolved in the lipid bilayer. Electron carriers 1 through 5 are each
bound in a specific position on a 596-amino-acid
trans-membrane protein formed from two separate subunits (see Figure 10-33).
Following excitation by a photon of light, a high-energy electron passes
from pigment molecule
to pigment molecule, very rapidly creating a charge separation as shown in the
sequence in steps A through D above, where the pigment molecule carrying
high-energy
electrons is colored green. Step E occurs more slowly. Once
released into the bilayer, the
quinone with two electrons loses its charge by picking up two protons (see Figure 14-31).
. As
outlined previously (Figure 14-48), in
a reaction center, light causes a net electron transfer from a weak electron
donor to a molecule that is a strong electron donor in its reduced form. In
this way
the excitation energy that would otherwise be released as fluorescence or heat
or both is used instead to raise the energy of an electron and thereby
create a
strong electron donor where none had been before. In this bacterium the weak
electron donor is a cytochrome (orange box), and the strong
electron donor is a
quinone (yellow
box). In the chloroplasts of higher
plants, as we discuss later, water,
rather than cytochrome, serves as the initial electron donor, which is why
oxygen
is released by photosynthesis in plants.Photosynthesis in plants and cyanobacteria produces both ATP and NADPH directly by a two-step process called noncyclic photophosphorylation. Because two photosystems 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.
In the first of the two photosystems - called photosystem II for historical reasons - the oxygens of two water molecules bind to a cluster of manganese atoms in a poorly understood water-splitting enzyme that enables electrons to be removed one at a time to fill the 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+ + 4 e - + O2.
): all three complexes accept electrons from
quinones and
pump H+ across the membrane. Note that the
H+ released by water oxidation and the
H+ taken up during NADPH formation also contribute to
the generation of the electrochemical
H+ gradient, which drives ATP synthesis by an ATP synthase
present in
this same membrane (not shown).
.
This Z scheme for ATP production is called noncyclic photophosphorylation to distinguish it from
a cyclic scheme that utilizes only photosystem I (see the text).
and 14-52).
The final electron acceptor in this electron-transport chain is the second
photosystem (photosystem I), which accepts an electron into the
hole created by light
in the chlorophyll molecule in its reaction center. Each electron that enters
photosystem I is 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; this last step also involves the
uptake of a
H+from the medium (Figure 14-52).
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 more 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 probably the energy change required to pass an electron efficiently from
water to NADP+. The use of two separate photosystems in
series also means that
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), which allows ATP synthase to harness
some of
the light-derived energy for ATP production.
). 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 back to the b6-f complex rather than being
passed on to
NADP+, and the electron is then recycled 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 to increase the
electrochemical proton gradient that drives the ATP synthase.
In summary, cyclic photophosphorylation involves only photosystem I, and it produces ATP without the formation of either NADPH or O2. Thus the relative activities of cyclic and noncyclic electron flows can determine how much light energy is converted into reducing power (NADPH) and how much into high-energy phosphate bonds (ATP).
Those compartments with a similar pH have been colored similarly. The proton-motive force across the thylakoid membrane consists almost entirely of the pH gradient; a high permeability of this membrane to Mg2+ and Cl- ions allows the flow of these ions to dissipate most of the membrane potential. Mitochondria presumably need a large membrane potential because they could not tolerate having their matrix at pH 10, as would be required to generate their proton-motive force without one.
, in chloroplasts H+ is pumped
out of the stroma (pH 8) into the thylakoid
space (pH about 5), creating a gradient of 3 to 3.5 pH units. This
represents a
proton-motive force of about 200 mV across the thylakoid membrane (nearly
all of
which is contributed by the pH gradient rather than by a membrane potential),
which drives ATP synthesis by the ATP synthase embedded in this membrane.
).
Although there are many similarities between mitochondria and chloroplasts, the structure of chloroplasts makes their electron- and proton-transport processes easier to study: by breaking both the inner and outer membranes of a chloroplast, isolated thylakoid discs can be obtained intact. These thylakoids resemble submitochondrial particles in that they have a membrane whose electron-transport chain has its utilization sites for NADP+, ADP, and phosphate all freely accessible to the outside. But isolated thylakoids retain their undisturbed native structure and are much more active than isolated submitochondrial particles. For this reason several of the experiments that first demonstrated the central role of chemiosmotic mechanisms were carried out with chloroplasts rather than with mitochondria.
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 inorganic phosphate.
.) 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.The chloroplast carries out 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.
Chloroplasts and photosynthetic bacteria obtain high-energy electrons by means of photosystems that capture the electrons excited when sunlight is absorbed by chlorophyll molecules. Photosystems are composed of an antenna complex attached to a photochemical reaction center, which is a precisely ordered complex of proteins and pigments in which the photochemistry of photosynthesis occurs. By far the best-understood photochemical reaction center is that of the purple photosynthetic bacteria, for which the complete three-dimensional structure is known. Whereas these bacteria contain only a single photosystem, there are two photosystems in chloroplasts and cyanobacteria. The two photosystems are normally linked in series and transfer electrons from water to NADP+ to form NADPH, with the concomitant production of a transmembrane electrochemical proton gradient; molecular oxygen (O2) is generated as a by-product.
Compared to mitochondria, chloroplasts have an additional internal membrane (the thylakoid membrane) and 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 other 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.
Much of the structure, function, and evolution of cells and organisms can be related to their need for energy. We have seen that the fundamental mechanisms for harnessing energy from such disparate sources as light and the oxidation of glucose are the same. Apparently, an effective method for synthesizing ATP arose early in evolution and has since been conserved with only small variations. How did the crucial individual components - ATP synthase, redox-driven H+ pumps, and photosystems - first arise? Hypotheses about events occurring on an evolutionary time scale are difficult to test. But clues abound, both in the many different primitive electron-transport chains that survive in some present-day bacteria and in geological evidence concerning the environment of the earth billions of years ago.
As explained in Chapter 1, the first living cells are thought to have arisen more than 3.5 x 109 years ago, when the earth was not more than about 109 years old. Because the environment lacked oxygen but was rich in geochemically produced organic molecules, the earliest metabolic pathways for producing ATP presumably 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 2-14). Without O2 to serve as the final electron acceptor, the electrons lost from the oxidized organic molecules must be 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 is excreted into the medium as a metabolic waste product; others, such as pyruvate, are retained by the cell for biosynthesis.
The end products are highlighted by green boxes. In both cases two molecules of NAD+ are used for each molecule of glucose that undergoes glycolysis, and these are regenerated by the transfer of hydride ions from NADH. In (A) the hydride ions are transferred to pyruvate to produce two molecules of lactic acid, which is excreted. In (B) the two hydride ions are successively transferred from two NADH molecules to compounds derived from pyruvate that produce succinic acid; for each molecule of succinic acid excreted, a molecule of pyruvate (red) is saved for biosyntheses inside the cell. In both (A) and (B) an organic acid must be excreted to re-form NAD+ and thereby enable glycolysis to continue in the absence of oxygen.
.The early fermentation processes would have provided not only the ATP but also the reducing power (as NADH or NADPH) required for essential biosyntheses, and many of the major metabolic pathways probably 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:
The continuous excretion of organic acids lowered the pH of the environment, favoring the evolution of proteins that function as transmembrane H+ pumps that could 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.
In such anaerobic bacteria, including E. coli, the oxidation is mediated by an energy-conserving electron-transport chain in the plasma membrane. As indicated, the starting materials are formic acid and fumarate, and the products are succinate and CO2. Note that H+ is consumed inside the cell and generated outside the cell, which is equivalent to pumping H+ to the cell exterior. Thus this membrane-bound electron-transport system can generate an electrochemical proton gradient across the plasma membrane. The redox potential of the formic-acid-CO2 pair is -420 mV, while that of the fumarate-succinate pair is +30 mV.
These pathways generate all the cell's ATP and reducing power from the oxidation of inorganic molecules, such as iron, ammonia, nitrite, and sulfur compounds. As indicated, some species can grow anaerobically by substituting nitrate for oxygen as the terminal electron acceptor. Most use the carbon-fixation cycle and synthesize their organic molecules entirely from carbon dioxide. The forward electron flows cause H+ to be pumped out of the cell, and the resulting H+gradient drives the production of ATP by an ATP synthase (not shown). The NADPH required for carbon fixation is produced by a reverse electron flow that is also driven by the H+gradient, as indicated.
). Others have similar
electron-transport components devoted solely to the oxidation and
reduction of inorganic substrates (see Figure 14-57, for example).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.
One possible sequence is shown.
.
). 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.
The photosystem in green bacteria resembles photosystem I in plants and cyanobacteria in using a series of iron-sulfur centers as primary electron acceptors that eventually donate their high-energy electrons to ferredoxin (Fd).
). 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 - probably more than 3 x
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 14-58). Because the
electrons removed from H2S are at a much more negative redox
potential than those of
H2O (-230 mV compared to +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 next step, which is thought to have occurred with the development of the cyanobacteria at least 3 x 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 photo-system, 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 and therefore could spread and evolve in ways denied 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 measures 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.
As indicated, geological evidence suggests that there was more than a billion-year delay between the rise of cyanobacteria (thought to be the first organisms to release O2) and the time that high O2 levels began to accumulate in the atmosphere. This delay was probably due largely to the rich supply of dissolved ferrous iron in the oceans, which reacted with the released O2 to form enormous iron oxide deposits.
).
The availability of O2made 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 in-organic substrates could be transported to O2 as the terminal electron acceptor. Many present-day purple photosynthetic bacteria can switch between photosynthesis and respiration, depending on the availability of light and O2, by surprisingly minor reorganizations of their electron-transport chains.
Oxygen respiration is thought to have begun developing about 2 x 109 years ago. As indicated, it seems to have evolved independently in the green, purple, and blue-green (cyanobacterial) lines of photosynthetic bacteria. It is thought that an aerobic purple bacterium that had lost its ability to photosynthesize gave rise to the mitochondrion, while several different blue-green bacteria gave rise to chloroplasts. Nucleotide sequence analyses suggest that mitochondria arose from bacteria that resembled the rhizobacteria, agrobacteria, and rickettsias - three closely related species known to form intimate associations with present-day eucaryotic cells.
outlines some of the
suspected evolutionary pathways just
discussed.
Bacteria, chloroplasts, and mitochondria all contain a membrane-bound enzyme complex that closely resembles the cytochrome b-c1 complex of mitochondria. These complexes all accept electrons from a quinone carrier (designated as Q) and pump H+ across their respective membranes. Moreover, in reconstituted in vitro systems the different complexes can substitute for one another, and the amino acid sequences of their protein components reveal that they are evolutionarily related.
).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 probably 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. 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 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 CO2 to 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 billion years ago, the O2 released by photosynthesis in cyanobacteria began to accumulate in the atmosphere. Once both organic molecules and O2 were 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 in the mitochondria of eucaryotes, and there is considerable evidence that both mitochondria and chloroplasts evolved from aerobic bacteria that were endocytosed by primitive eucaryotic cells.
Cells must generate new cytoplasmic organelles if they are to grow and divide. They must also replenish organelles that are degraded as part of the continual process of organelle turnover in nonproliferating cells. Organelle biosynthesis requires the ordered synthesis of the requisite proteins and lipids and the delivery of each component to the correct organelle subcompartment. In Chapter 12we discussed how selected proteins and lipids are imported into mitochondria and chloroplasts from elsewhere in the cell. Here we describe the contributions that these energy-converting organelles make to their own biogenesis.
While most of the proteins in mitochondria and chloroplasts are encoded by nuclear DNA and imported into the organelle from the cytosol after they are synthesized on cytosolic ribosomes, some are encoded by organelle DNA and synthesized on ribosomes within the organelle. The protein traffic between the cytosol and these organelles seems to be unidirectional, as no protein is known to be exported from mitochondria or chloroplasts to the cytosol.
Each red arrow indicates the site of action of an inhibitor that is specific for either organelle or cytosolic protein synthesis.
). These inhibitors
are widely used in studies of the functions of these organelles.
.
(Courtesy of Daniel S. Friend.)
and 14-64),
implying that it is a controlled process rather than a chance pinching in two.
In most cells individual energy-converting organelles divide throughout interphase, out of phase with one another and with the division of the cell. Similarly, the replication of organelle DNA is not limited to the S phase, when nuclear DNA replicates, but occurs throughout the cell cycle. 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, so that each cell type maintains a constant amount of organelle DNA.
The number of organelles 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. Moreover, in special circumstances, organelle division is precisely controlled by the cell: thus, in some algae that contain only one or a few chloroplasts, the organelle divides just prior to cytokinesis in a plane that is identical to the future plane of cell division.
| Type of DNA | Size (thousands of nucleotide pairs) |
|---|---|
| Chloroplast DNA | |
| Higher plants | 120-200 |
| Chlamydomonas(green alga) | 180 |
| Mitochondrial DNA | |
| Animals (including flatworms, insects, and mammals) | 16-19 |
| Higher plants | 150-2500 |
| Fungi | |
 Schizosaccharomyces pombe(fission yeast) | 17 |
| Aspergillus nidulans | 32 |
| Neurospora crassa | 60 |
| Saccharomyces cerevisiae(budding yeast) | 78 |
| Chlamydomonas(green alga) | 16 (linear molecule) |
| Protozoa | |
| Trypanosoma brucei | 22 |
| Paramecium | 40 (linear molecule) |
These genomes are circular DNA molecules unless indicated otherwise.
The circular DNA genome has replicated only between the two points marked by arrows (yellow strands). (Courtesy of David Clayton.)
). Plants, however, contain a circular
mitochondrial genome that is 10 to 150
times larger, depending on the plant. The largest of these are about half
the size of
typical bacterial genomes, which are also circular DNA molecules.
Despite the small number of proteins encoded in their genomes, mitochondria and plastids carry out their own DNA replication, DNA transcription, and protein synthesis. These processes take place in the matrix in mitochondria and in the stroma in 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 the case of chloroplasts:
Chloroplast ribosomes are very similar to E. coli ribosomes, both in their sensitivity to various antibiotics (such as chloramphenicol, streptomycin, erythromycin, and tetracycline) and in their structure. Not only are the nucleotide sequences of the ribosomal RNAs of chloroplasts and E. coli strikingly similar, but chloroplast ribosomes are able to use bacterial tRNAs in protein synthesis. In all these respects, chloroplast ribosomes differ from those found in the cytosol of the same plant cell.
Protein synthesis in chloroplasts starts with N-formylmethionine, as in bacteria, and not with methionine, as in the cytosol of eucaryotic cells.
Unlike nuclear DNA, chloroplast DNA can be transcribed by the RNA polymerase enzyme from E. coli to produce chloroplast mRNAs, and these mRNAs are efficiently translated by an E. coli protein-synthesizing system.
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-formylmethionine.
The complete nucleotide sequence of this genome has been determined. The organization of the chloroplast genome is very similar in all higher plants, although the size varies from species to species depending on how much of the DNA surrounding the genes encoding the chloroplast's 16S and 23S ribosomal RNAs is present in two copies.
). In
addition, the DNA sequences present
seem to encode at least 40 proteins whose functions are unknown.
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.
Possible reasons will be discussed 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. Protein sequences encoded in chloroplasts are clearly recognizable as bacterial, and several clusters of genes with related functions (for example, those encoding ribosomal proteins) are organized in the same way in the genomes of chloroplasts, E. coli, and cyanobacteria.
Detailed comparisons of large numbers of homologous nucleotide sequences should help to 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 transferred and stably maintained. In higher plants, for example, two-thirds of the 60 or so chloroplast ribosomal proteins are encoded in the cell nucleus, although the genes have a clear bacterial ancestry and the chloroplast ribosomes retain their original bacterial properties.
The genome contains 2 rRNA genes, 22 tRNA genes, and 13 protein-coding sequences. The DNAs of several other animal mitochondrial genomes have also been completely sequenced and have the same genes and gene organization.
).
Compared to nuclear, chloroplast, and bacterial genomes, the human mitochondrial genome has several surprising features. (1) Unlike other genomes, nearly every nucleotide appears 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) 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) Perhaps most surprising, comparison of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicates that the genetic code is different, so that 4 of the 64 codons have different "meanings" from those of the same codons in other genomes (Table 14-4).
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, 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 carry out these processes are relatively simple compared to 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 mitochondrial DNA sequence comparisons especially useful for estimating the dates of relatively recent evolutionary events, such as the steps in primate development.
Mitochondrial genomes are much larger in plant than in animal cells, and they vary remarkably in their DNA content, ranging from about 150,000 to about 2.5 x 106 nucleotide pairs. Yet these genomes seem to encode only a few more proteins than do animal mitochondrial genomes. The paradox is compounded by the observation that in one family of plants, the cucurbits, mitochondrial genomes vary in size by as much as sevenfold. The green alga Chlamydomonas has a linear mitochondrial genome of only 16,000 nucleotide pairs, the same size as in animals.
Although very little sequence information is available for higher plant mitochondrial DNA molecules, almost all of the 70,000 nucleotide pairs in the large mitochondrial genome of the yeast Saccharomyces cerevisiae have been sequenced, and only about one-third of them code for protein. This finding raises the possibility that much of the extra DNA in yeast mitochondria, and possibly in plant mitochondria as well, is "junk DNA" of little consequence to the organism.
The processing of precursor RNAs plays 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 - called the heavy strand (H strand) because of its density in CsCl - 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 light strand (L strand) transcript is processed to produce only eight tRNAs and one 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 about 20 plant chloroplast genes. Many of the introns in organelle genes consist of related nucleotide sequences that are capable of splicing themselves out of the RNA transcripts by RNA-mediated catalysis (see p. 109), 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. On the other hand, introns in other yeast mitochondrial genes have 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 seems likely that the intron sequences themselves are of ancient origin and that, while they have been lost from many bacteria, they have been preferentially retained in those organelle genomes where RNA splicing is regulated to help control gene expression.
Most experiments on the mechanisms of mitochondrial biogenesis have been performed with Saccharomyces cerevisiae (baker's yeast). There are several reasons for this. First, when grown on glucose, this yeast has an ability to live by glycolysis alone and can therefore survive without functional mitochondria, which are required for oxidative phosphorylation. This makes it possible to grow cells with mutations in mitochondrial or nuclear DNA that drastically interfere with mitochondrial biogenesis; 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 (asymmetrical mitosis), 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. Because mutations in mitochondrial genes are not inherited according to the Mendelian rules that govern the inheritance of nuclear genes, genetic studies reveal which of the genes involved in mitochondrial function are located in the nucleus and which in the mitochondria.
For each nuclear gene two of the four cells that result from meiosis inherit the gene from one of the original haploid parent cells and the remaining two cells inherit the gene from the other (Mendelian inheritance). In contrast, because of the gradual mitotic segregation of mitochondria during vegetative growth (see text), it is possible for all four of the cells that result from meiosis to inherit their mitochondrial genes from only one of the two original haploid cells (non-Mendelian, or cytoplasmic, inheritance). In this example the mitochondrial gene is one that (in its mutant form) makes protein synthesis in the mitochondrion resistant to chloramphenicol, a protein synthesis inhibitor that acts specifically on energy-converting organelles and bacteria. Yeast cells that contain the mutant gene can be detected by their ability to grow in the presence of chloramphenicol on a substrate, such as glycerol, that cannot be used for glycolysis. With glycolysis blocked, ATP must be provided by functional mitochondria, and therefore only cells that carry chloramphenicol-resistant mitochondria will grow.
. 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 will contain a mixture of mutant and wild-type
mitochondria.
But when the zygote undergoes mitosis to produce a diploid daughter, the
mutant and wild-type mitochondria will be distributed at random between the
mother and the daughter cell, so that each daughter is likely to inherit
more mutant
or more wild-type mitochondria. With successive mitotic divisions, either the
mutant or the wild-type mitochondria will gradually be diluted out of some
daughters by the same random process, leaving mitochondria of only one type.
Thereafter, all of the progeny from that daughter will have mitochondria that
are genetically identical. Thus this random process, called mitotic segregation, will eventually produce diploid yeast cells
with only a single type of
mitochondrial DNA. When such diploid cells 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, to contrast it with
the Mendelian inheritance of nuclear genes (see Figure 14-68). When it occurs,
it demonstrates that the gene in question is located outside the nuclear
chromosomes and therefore probably in the yeast mitochondria.
). 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 expect
mitochondrial inheritance in higher animals, therefore, 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, human mitochondrial DNA has
been shown to be 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.
Genetic studies of yeasts have played a crucial part 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.
In petite mutants all of the mitochondrion-encoded gene products are missing, and so the organelle is constructed entirely from nucleus-encoded proteins. (Courtesy of Barbara Stevens.)
), and they
contain virtually all of 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.
The 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. Many biologists believe that peroxisomes
normally replicate in this way (see Figure 12-29).
For chloroplasts the nearest equivalent to yeast mitochondrial petite mutants are mutants of unicellular algae such as Euglena. Cells 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 as soon as their food stores run out.
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. Nuclear-encoded enzymes in the mitochondrial matrix carry out 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. In addition, the respiratory enzyme complexes in the mitochondrial inner membrane of mammals contain several tissue-specific, nuclear-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 nuclear-encoded proteins in chloroplasts.
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, on the other hand, 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; it is found mainly in the mitochondrial inner membrane, where it constitutes about 20% of the total lipid.
We have discussed the important question of how specific cytosolic proteins are imported into mitochondria and chloroplasts in detail in Chapter 12.
Microsporidia and Giardia are two present-day anaerobic single-celled eucaryotes (protozoa) without mitochondria. Because they have an rRNA sequence that suggests a great deal of evolutionary distance from all other known eucaryotes, it has been postulated that their ancestors were also anaerobic and resembled the eucaryote that first engulfed the precursors of mitochondria.
). 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
x 109 years ago, before animals and plants separated (see Figure 14-59). Plant and
algal chloroplasts seem to have been derived later from an
endocytic event
involving an oxygen-evolving photosynthetic bacterium. In order 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 separate events
of this
kind occurred.
Since most of the genes encoding present-day mitochondrial and chloroplast proteins are in the cell nucleus, it seems that an extensive transfer of genes from organelle to nuclear DNA has occurred during eucaryote evolution. This would explain why some of the nuclear genes encoding mitochondrial 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 cells. 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."
. It appears that
mitochondria are descendants of
a particular type of purple photosynthetic bacterium that had previously lost
its ability to carry out photosynthesis and was left with only a
respiratory chain.
It is not clear that all mitochondria have originated from a single
endosymbiotic event, however. While the mitochondria from protozoans have
distinctly procaryotic features, for example, some of them are sufficiently
different
from plant and animal mitochondria to suggest a separate origin.
The proteins imported from the cytosol play a major part in creating the genetic system of the mitochondrion in addition to contributing most of the organelle protein. The mitochondrion itself contributes only mRNAs, rRNAs, and tRNAs to its genetic system. Not indicated in this diagram are the additional nucleus-encoded proteins that regulate the expression of individual mitochondrial genes at posttranscriptional levels.
). 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 there is reason to think 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
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 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. 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, in the case of mitochondria, by recent alterations in the mitochondrial genetic code that made the remaining mitochondrial genes nonfunctional if they were transferred to the nucleus.
Mitochondria and chloroplasts grow and divide in two in a coordinated process that requires the contribution of two separate genetic systems - that of the organelle and that of 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 partially functional organelles will 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 bacterial-like enzymes that are synthesized on cytosolic ribosomes and then imported into the organelle.
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