Proton diffusion is coupled to ATP synthesis

As we have seen, all the carriers and enzymes of the respiratory chain except cytochrome c are embedded in the inner mitochondrial membrane. The operation of the respiratory chain results in the active transport of protons (H+), against their concentration gradient, across the inner membrane of the mitochondrion from the mitochondrial matrix to the intermembrane space (the space between the inner and outer mitochondrial membranes). This occurs because the electron carriers contained in the three large protein complexes are arranged such that protons are taken up on one side of the membrane (the mitochondrial matrix) and transported along with electrons to the other side (the intermembrane space). Thus, the protein complexes act as proton pumps. Because of the positive charge on the protons (H+), this transport causes not only a difference in proton concentration, but also a difference in electric charge, across the membrane, with the inside of the organelle (the matrix) more negative than the intermembrane space.

Together, the proton concentration gradient and the charge difference constitute a source of potential energy called the proton-motive force. This force tends to drive the protons back across the membrane, just as the charge on a battery drives the flow of electrons discharging the battery. The conversion of the proton-motive force into kinetic energy is prevented by the fact that protons cannot cross the hydrophobic lipid bilayer of the inner membrane by simple diffusion.

However, they can diffuse across the membrane by passing through a specific proton channel, called ATP synthase, that couples proton movement to the synthesis of ATP. This coupling of proton-motive force and ATP synthesis is called the chemiosmotic mechanism, or chemiosmosis.

The chemiosmotic mechanism for ATP synthesis

The chemiosmotic mechanism uses ATP synthase to couple proton diffusion to ATP synthesis. This mechanism has three parts:

1. The flow of electrons from one electron carrier to another in the respiratory chain is a series of exergonic reactions that occurs in the inner mitochondrial membrane.

2. These exergonic reactions drive the endergonic pumping of H+ out of the mitochondrial matrix and across the inner membrane into the intermembrane space. This pumping establishes and maintains a H+ gradient.

3. The potential energy of the H+ gradient or protonmotive force, is harnessed by ATP synthase. This protein has two roles: It acts as a channel allowing the H+ to diffuse back into the matrix and it uses the energy of that diffusion to make ATP from ADP and Pi. ATP synthesis is a reversible reaction and ATP synthase can also act as an ATPase, hydrolyzing ATP to ADP and Pi: ATP®ADP + Pi + free energy

If the reaction goes to the right, free energy is released, and that energy is used to pump H+ out of the mitochondrial matrix. If the reaction goes to the left, it uses free energy from H+ diffusion into the matrix to make ATP. What makes it prefer ATP synthesis? There are two answers to this question.

ATP leaves the mitochondrial matrix for use elsewhere in the cell as soon as it is made, keeping the ATP concentration in the matrix low and driving the reaction toward the left. A person hydrolyzes about 1025 ATP molecules per day, and clearly the vast majority are recycled using the free energy from the oxidation of glucose.

The H+ gradient is maintained by electron transport and proton pumping. (The electrons, you will recall, come from the oxidation of NADH and FADH₂, which are themselves reduced by the oxidations of glycolysis and the citric acid cycle. Thus, one reason you eat is to replenish the H+ gradient!) ATP synthase is a large multi-protein machine, containing 16 different polypeptides in mammals. It has two functional components. One of these components is the membrane channel for H+. The other component sticks out into the mitochondrial matrix like a lollipop and contains the active site for ATP synthesis. The actual mechanism of transferring energy from the H+ gradient to the phosphorylation of ADP involves the physical rotation of the core of the enzyme, with this rotational energy transferred to ATP.

Experiments demonstrate chemiosmosis

Two key experiments demonstrated (1) that a proton (H+) gradi ent across a membrane can drive ATP synthesis; and (2) that the enzyme ATP synthase is the catalyst for this reaction.

Experiment 1 tested the hypothesis that ATP synthesis is driven by the H+ gradient across an inner mitochondrial membrane. In this experiment, mitochondria without a food source were “fooled” into making ATP when researchers raised the H+ concentration in their environment. A sample of isolated mitochondria that had been exposed to a low H+ concentration was suddenly put in a medium with a high concentration of H+. The outer mitochondrial membrane, unlike the inner one, is freely permeable to H+, so H+ rapidly diffused into the intermembrane space. This created an artificial gradient across the inner membrane, which the mitochondria used to make ATP from ADP and Pi. This result supported the hypothesis and provided strong evidence for chemiosmosis.

Experiment 2 tested the hypothesis that the enzyme ATPase couples a proton gradient to ATP synthesis. In this experiment, a proton pump isolated from a bacterium was added to artificial membrane vesicles. When an appropriate energy source was provided, H+ was pumped into the vesicles, creating a gradient. If mammalian ATP synthase was then inserted into the membranes of these vesicles and the energy source removed, the vesicles made ATP even in the absence of the usual electron carriers. Again, the result supported the hypothesis, showing that the enzyme ATP synthase is the coupling factor in the membrane.

Uncoupling proton diffusion from ATP production

For the chemiosmotic mechanism to work, the diffusion of H+ and the formation of ATP must be tightly coupled; that is, the protons must pass only through the ATP synthase channel in order to move into the mitochondrial matrix. If a second type of H+ diffusion channel (not ATP synthase) is inserted into the mitochondrial membrane, the energy of the H+ gradient is released as heat, rather than being coupled to the synthesis of ATP. Such uncoupling molecules are deliberately used by some organisms to generate heat instead of ATP. For example, the natural uncoupling protein thermogenin plays an important role in regulating the temperature of some mammals, especially newborn human infants who lack the hair to keep warm and of hibernating animals.

Fermentation: ATP from Glucose, without O₂

Recall that fermentation is the breakdown of the pyruvate produced by glycolysis in the absence of O₂. Because fermentation results in the incomplete oxidation of glucose, it releases much less energy than cellular respiration. Why would such an inefficient process exist? Suppose the supply of oxygen to a respiring cell is reduced (an anaerobic condition). As a consequence, oxygen is no longer available to pick up electrons at the end of the respiratory chain. As we can deduce from Figure , the first consequence of an insufficient supply of O2 is that the cell cannot reoxidize cytochrome c, so all of that compound is soon in the reduced form. When this happens, QH₂ cannot be oxidized back to Q, and soon all the Q is in the reduced form. So it goes, until the entire respiratory chain is reduced. Under these circumstances, no NAD+ and no FAD are regenerated from their reduced forms. Therefore, the oxidative steps in glycolysis, pyruvate oxidation, and the citric acid cycle also stop. If the cell has no other way to obtain energy from its food, it will die. Under anaerobic conditions, many (but not all) cells can produce a small amount of ATP by glycolysis, provided that fermentation metabolizes and regenerates the NAD+ necessary to keep glycolysis running. Fermentation, like glycolysis, occurs in the cytoplasm. It has two defining characteristics:

_ Fermentation uses NADH + H+ formed by glycolysis to reduce pyruvate or one of its metabolites, and consequently NAD+ is regenerated. NAD+ is required for reaction 6 of glycolysis, so once the cell has replenished its NAD+ supply in this way, it can carry more glucose through glycolysis.

_ Fermentation enables glycolysis to produce a small but sustained amount of ATP. The reactions of fermentation do not themselves produce any ATP. Only as much ATP is produced as can be obtained from substrate-level phosphorylation—not the much greater yield of ATP obtained by cellular respiration using chemiosmosis.

When cells capable of fermentation become anaerobic, the rate of glycolysis speeds up tenfold or even more. Thus a substantial rate of ATP production is maintained, although efficiency in terms of ATP molecules per glucose molecule is greatly reduced compared with cellular respiration under aerobic conditions. Some organisms are confined to totally anaerobic environments and use only fermentation. Usually, there are two metabolic reasons for this. First, these organisms lack the molecular machinery for oxidative phosphorylation, and second, they lack enzymes to detoxify the toxic by-products of O2, such as hydrogen peroxide (H₂O₂). An example of such an obligate anaerobe is Clostridium botulinum, the bacterium that thrives in sealed containers of foods and releases the potentially deadly botulinum toxin. Other bacteria, such as Mycobacterium tuberculosis, which causes tuberculosis, cannot carry out fermentation and must grow in aerobic environments. Still others, such as Escherichia coli, which grows in the human large intestine, can perform either respiration or fermentation, but prefer the former in an aerobic environment. And several bacteria carry on cellular respiration—not fermentation— without using oxygen gas as an electron acceptor. Instead, to oxidize their cytochromes, these bacteria reduce nitrate ions (NO₃ ^–) to nitrite ions (NO₂^–).

Some fermenting cells produce lactic acid and some produce alcohol

Different types of fermentation are carried out by different bacteria and eukaryotic body cells. These different fermentation processes are distinguished by the final product produced. For example, in lactic acid fermentation, pyruvate is reduced to lactate. Lactic acid fermentation takes place in many microorganisms as well as in our muscle cells. Unlike muscle cells, nerve cells (neurons) are incapable of fermentation because they lack the enzyme that reduces pyruvate to lactate. For that reason, without adequate oxygen the human nervous system (including the brain) is rapidly destroyed; it is the first part of the body to die. Certain yeasts and some plant cells carry on a process called alcoholic fermentation under anaerobic conditions (Figure ). This process requires two enzymes to metabolize pyruvate. First, carbon dioxide is removed from pyruvate, leaving the compound acetaldehyde. Second, the acetaldehyde is reduced by NADH + H+, producing NAD+ and ethyl alcohol (ethanol). This is how beer and wine are made.

Contrasting Energy Yields

The total net energy yield from glycolysis using fermentation is two molecules of ATP per molecule of glucose oxidized. In contrast, the maximum yield that can be obtained from a molecule of glucose through glycolysis followed by cellular respiration is much greater —about 36 molecules of ATP.

Why is so much more ATP produced by cellular respiration? As we have repeatedly stated, glycolysis is only a partial oxidation of glucose, as is fermentation. Much more energy remains in the end products of fermentation, such as lactic acid and ethanol, than in the end product of cellular respiration, CO₂. In cellular respiration, carriers (mostly NAD+) are reduced in pyruvate oxidation and the citric acid cycle, then oxidized by the respiratory chain, with the accompanying production of ATP (three for each NADH + H+ and two for each FADH₂) by the chemiosmotic mechanism. In an aerobic environment, an organism capable of this type of metabolism will be at an advantage (in terms of energy availability per glucose molecule) over one limited to fermentation. The total gross yield of ATP from one molecule of glucose processed through glycolysis and cellular respiration is 38. However, we may subtract two from that gross — for a net yield of 36 ATP — because in some animal cells the inner mitochondrial membrane is impermeable to NADH and a “toll” of one ATP must be paid for each NADH produced in glycolysis that is shuttled into the mitochondrial matrix.

Relationships between Metabolic Pathways

Glycolysis and the pathways of cellular respiration do not operate in isolation from the rest of metabolism. Rather, there is an interchange, with biochemical traffic flowing both into these pathways and out of them, to and from the synthesis and breakdown of amino acids, nucleotides, fatty acids, and so forth. Carbon skeletons enter from other molecules that are broken down to release their energy (catabolism), and carbon skeletons leave to form the major macromolecular constituents of the cell (anabolism).

Catabolism and anabolism involve interconversions using carbon skeletons

A hamburger or veggiburger contains three major sources of carbon skeletons for the person who eats it: carbohydrates, mostly as starch (a polysaccharide); lipids, mostly as triglycerides (three fatty acids attached to glycerol); and proteins (polymers of amino acids).

Catabolic interconversions

Polysaccharides, lipids, and proteins can all be broken down to provide energy:

Polysaccharides are hydrolyzed to glucose. Glucose then passes through glycolysis and the citric acid cycle, where its energy is captured in NADH and ATP.

Lipids are broken down into their substituents, glycerol and fatty acids. Glycerol is converted to dihydroxyacetone phosphate, an intermediate in glycolysis, and fatty acids are converted to acetate and then acetyl CoA in the mitochondria. In both cases, further oxidation to CO₂ and release of energy then occur.

Proteins are hydrolyzed to their amino acid building blocks. The 20 different amino acids feed into glycolysis or the citric acid cycle at different points.

Catabolism and anabolism are integrated

A carbon atom from a protein in your burger can end up in DNA or fat or CO₂, among other fates. How does the cell “decide” which metabolic pathway to follow? With all of these possible interconversions, you might expect that the cellular concentrations of various biochemical molecules would vary widely. For example, the level of oxaloacetate in your cells might depend on what you eat (some food molecules form oxaloacetate) and whether oxaloacetate is used up (in the citric acid cycle or in forming the amino acid aspartate). Remarkably, the levels of these substances in what is called the “metabolic pool” — the sum total of all the small molecules such as metabolic intermediates in a cell — are quite constant. The cell regulates the enzymes of catabolism and anabolism so as to maintain a balance. This metabolic homeostasis gets upset only in unusual circumstances. Let’s look one such unusual circumstance: undernutrition. Glucose is an excellent source of energy. The fats and proteins can also serve as energy sources. Any one, or all three, could be used to provide the energy your body needs. In reality, things are not so simple. Proteins, for example, have essential roles in your body as enzymes and structural elements, and using them for energy might deprive you of a catalyst for a vital reaction. Polysaccharides and fats have no such catalytic roles. But polysaccharides, because they are somewhat polar, can bind a lot of water. Because they are nonpolar, fats do not bind as much water as polysaccharides do. Thus, in water, fats weigh less than polysaccharides. Also, fats are more reduced than carbohydrates (more C—H bonds as opposed to C—OH) and have more energy stored in their bonds. For these two reasons, fats are a better way for an organism to store energy than polysaccharides. It is not surprising, then, that a typical person has about one day’s worth of food energy stored as glycogen, a week’s food energy as usable proteins in blood and over a month’s food energy stored as fats. What happens if a person does not eat enough food to produce sufficient ATP and NADH for anabolism and biological activities? This situation can be the result of a deliberate decision to lose weight, but for too many people, it is forced upon them because not enough food is available. In either case, the first energy stores in the body to be used are the glycogen stores in muscle and liver cells. This doesn’t last long, and next come the fats. The level of acetyl CoA rises as fatty acids are broken down. However, a problem remains: Because fatty acids cannot get from the blood to the brain, the brain can use only glucose as its energy source. With glucose already depleted, the body must convert something else to make glucose for the brain. This gluconeogenesis uses mostly amino acids, largely from the breakdown of proteins. So, without sufficient food intake, both proteins (for glucose) and fats (for energy) are used up. After several weeks of starvation, fat stores become depleted, and the only energy source left is proteins, some of which have already been degraded to supply the brain with glucose. At this point, essential proteins, such as antibodies used to fight off infections and muscle proteins, get broken down, both for energy and for gluconeogenesis. The loss of these proteins can lead to severe illnesses.

Date: 2014-12-22; view: 961