Respiration in the cells

Plant cells are alive, of course, so they use up food for energy. The green plants can provide you with some of the energy you need to live. First you must get this energy to your cells where it can be used. But what happens to food inside the cell? It is broken down, or changed. Respiration – the step in metabolism in which food is broken down - takes place in the mitochondria.

In respiration, food is broken down to give off energy. But the food materials are not broken down all at once. Instead, they are broken down in a series of small steps. Energy is given off with each step (Chart 2.1).

Some of the food materials used by the cell are carbohydrates. Carbohydrates, such as sugars and starches, made up of large molecules, must be broken down into smaller molecules in the mitochondria.

Breaking down molecules means dividing them into smaller, simpler molecules. The smaller, simpler molecules are molecules of materials that are different from the original materials. Energy is given off each time the molecules are divided. Finally, the remaining molecules cannot be divided any more.

In general, the cells in living things break down food molecules to form molecules of water and molecules of a gas called carbon dioxide. The energy given off in breaking down food is used for the life function of the cells.

Respiration occurs during both light and dark hours. On a warm sunny day, plants produce much more food and oxygen through photosynthesis than they can use. At night, plants use up some of this excess food for respiration.

Due to the types of respiration, all the organisms are divided in for:

Aerobic (air + bios – life): requiring free oxygen for respiration.

Anaerobic: applied to the cells (mostly bacterial) that can live without free oxygen; obligate anaerobes cannot live in the presence of oxygen; facultative anaerobes can live with or without oxygen.

Chart 2.1

Comparison of photosynthesis and respiration.

Photosynthesis	Respiration
Occurs only in the presence of chlorophyll & light	Occurs in all cells & goes on day & night
Energy is stored in sugars	Energy is released from sugars
Carbon dioxide & water are used to make sugars	Carbon dioxide & water are produced
Oxygen is released	Oxygen is used

Glycolysis

The first step in cellular respiration is a process of glycolysis. In glycolysis, a glucose molecule is broken in half to form two three- carbon molecules of the substance called pyruvic acid.

Glycolysis takes place in the cytoplasm of the cell as well as in the mitochondria. The process involves a series of 9 enzyme-controlled reactions. Two reactions early in the process are endergonic; each requires the input of one molecule of ATP. The later reactions release enough energy to combine 4 molecules of ATP. Thus, glycolysis produces an overall gain of two molecules of ATP for each molecule of glucose.

In addition to producing two three- carbon molecules of pyruvic acid and two ATPs, glycolysis also releases 4 hydrogen atoms. These hydrogens then combine with a hydrogen acceptor, as in the light reactions of photosynthesis. The following equation (2.2) summarizes the overall reaction:

C₆H₁₂O₆  2C₂H₃OCOOH + 4H (2.2)

glucose pyruvic acid

Glycolysis is an anaerobic process—that is, no oxygen is required for the process to take place. Glycolysis is followed by one of two processes. If oxygen is present in the cell, the pyruvic acid is broken down further through the process of aerobic respiration. This process results in an additional gain of ATP molecules. Without oxygen, the pyruvic acid cannot be used to release more energy. However, the hydrogen acceptor molecules must release hydrogen atoms, othervise glycolysis could not continue. Hydrogen atoms are removed through fermentation, an anaerobic process that breaks down pyruvic acid into ethyl alcohol or lactic acid. Together, the processes of glycolysis and fermentation make up anaerobic respiration.

Fermentation

Fermentation occurs in some of the less complex organisms, such as some bacteria and yeasts. Most microorganisms convert pyruvic acid to ethyl alcohol, which some animal cells and other microorganisms convert into lactic acid.

Alcoholic fermentation combines the hydrogen and the pyruvic acid formed during glycolysis to produce ethyl alcohol.

Carbon dioxide is also given off as a byproduct. The general equation (2.3) of the process can be written:

2C₂H₃OCOOH + 4H  2C₂H₅OH + 2CO₂ (2.3)

pyruvic acid ethyl alcohol

Most of the energy originally stored in the glucose remains in the bonds of the ethyl alcohol molecule. For this reason, alcohol is a good fuel.

Lactic acid fermentation combines pyruvic acid and hydrogen from glycolysis to form lactic acid. The general equation for this process can be written (4.4):

2C₂H₃OCOOH + 4H  2CH₃CHOHCOOH (2.4)

pyruvic acid lactic acid

Lactic acid fermentation occurs in animal muscle cells. When oxygen is available, these cells carry on aerobic respiration. During strenuous exercise, oxygen concentration diminishes, and muscle cells are forced to use lactic acid fermentation. This process uses the hydrogen stored by the hydrogen acceptor molecule and makes the acceptor molecule available for reuse in glycolysis to obtain more energy. The accumulation of lactic acid in the cells is one of the causes of muscle soreness. When the oxygen concentration returns to normal, the lactic acid is converted back to pyruvic acid, aerobic respiration begins, and the soreness fades.

Aerobic Respiration

Like fermentation, aerobic respiration begins with the pyruvic acid produced through glycolysis. Although glycolysis takes place in the cytoplasm of the cell, aerobic respiration takes place on the folded membranes inside the mitochondria. Associated with these folded membranes are all the enzymes and coenzymes needed in the reactions that make up the process of aerobic respiration.

The equation for aerobic respiration (2.5) is essentially the reverse of that for photosynthesis:

enzymes

C₆H₁₂O₆ + 6O₂  6CO₂ + 6H₂O + energy (2.5)

Glucose

Aerobic respiration results in a maximum energy gain of 38 molecules of ATP from each molecule of glucose—that is, 36 ATPs are produced in the mitochondria in addition to the 2 gained from cytoplasmic glycolysis. Some cells, because of a high metabolic rate, must produce a greater amount of energy. Most species of living things carry on aerobic respiration because of its high energy yields. The highly active and complex organisms that inhabit the earth today could not have evolved without the energy provided by aerobic respiration.

The complete breakdown of one molecule of glucose results in a maximum of 38 molecules of ATP.

The same process of respiration that occurs in humans also occurs in the cells of all living things. Cabbage, mushrooms, and bacteria all carry out respiration.

The metabolism of sugars is important not only in making alcoholic beverages but in providing the energy that organisms store in ATP—the energy you use all the time to fuel both conscious actions such as turning the pages of this book and automatic ones such as the beating of the heart.

Energy and Electrons from Glucose

We are all familiar with fuels and their uses. Petroleum fuels contain stored energy that is harvested to move cars and heat homes. Wood burning in a stove or campfire releases energy as light and heat. Living organisms also need fuels, which must be obtained from foods. This is true whether we are speaking of organisms that make their own foods through photosynthesis or organisms that obtain foods by eating other organisms. The most common fuel for living cells is the sugar glucose (C6H12O6). Many other compounds serve as foods but almost all of them are converted to glucose or to intermediate compounds in the step-by-step metabolism of glucose. As you will see in this section, cells obtain energy from glucose by the chemical process of oxidation which is carried out through a series of metabolic pathways. Before we examine that process, let’s take a brief look at how metabolic pathways operate in the cell. Several principles govern metabolic pathways:

Complex chemical transformations in the cell do not occur in a single reaction, but in a number of separate reactions that form a metabolic pathway.

Each reaction in a pathway is catalyzed by a specific enzyme.

Metabolic pathways are similar in all organisms, from bacteria to humans.

Many metabolic pathways are compartmentalized in eukaryotes, with certain reactions occurring inside an organelle.

The operation of each metabolic pathway can be regulated by the activities of key enzymes.

Cells trap free energy while metabolizing glucose

The familiar process of combustion (burning) is very similar to the chemical processes that release energy in cells. If glucose is burned in a flame, it reacts with O₂, rapidly forming carbon dioxide and water and releasing a lot of energy. The balanced equation for this combustion reaction is C₆H₁₂O₆ + 6 O₂ 6 CO₂ + 6 H₂O + energy (heat and light).

The same equation applies to the metabolism of glucose in cells. The metabolism of glucose, however, is a multistep, controlled series of reactions. The multiple steps of the process permit about one-third of the energy released to be captured in ATP. That ATP can be used to do cellular work such as movement or active transport across a membrane, just as energy captured from combustion can be used to do work. The change in free energy (∆G) for the complete conversion of glucose and O₂ to CO₂ and water, whether by combustion or by metabolism, is –686 kcal/mol (–2,870 kJ/mol). Thus the overall reaction is highly exergonic and can drive the endergonic formation of a great deal of ATP from ADP and phosphate. It is the capture of this energy in ATP that requires the many steps characteristic of glucose metabolism. Three metabolic processes play roles in the utilization of glucose for energy: glycolysis, cellular respiration, and fermentation. All three involve metabolic pathways made up of many distinct chemical reactions.

Glycolysis begins glucose metabolism in all cells and produces two molecules of the three-carbon product pyruvate. Asmall amount of the energy stored in glucose is captured in usable forms. Glycolysis does not use O₂.

Cellular respiration uses O₂ from the environment and completely converts each pyruvate molecule to three molecules of CO₂ through a set of metabolic pathways. In the process, a great deal of the energy stored in the covalent bonds of pyruvate is released and transferred to ADP and phosphate to form ATP.

Fermentation does not involve O₂. Fermentation converts pyruvate into products such as lactic acid or ethyl alcohol (ethanol), which are still relatively energy-rich molecules. Because the breakdown of glucose is incomplete, much less energy is released by fermentation than by cellular respiration, and no ATP is produced. Glycolysis and fermentation are anaerobic metabolic processes — that is, they do not involve O₂. Cellular respiration is an aerobic metabolic process, requiring the direct participation of O₂.

Redox reactions transfer electrons and energy

In preceding section described the addition of phosphate groups to ADP to make ATP as an endergonic reaction that can extract and store energy from exergonic reactions. Another way of transferring energy is to transfer electrons. A reaction in which one substance transfers one or more electrons to another substance is called an oxidation–reduction reaction or redox reaction.

Reduction is the gain of one or more electrons by an atom, ion or molecule.

Oxidation is the loss of one or more electrons.

Although oxidation and reduction are always defined in terms of traffic in electrons, we may also think in these terms when hydrogen atoms (not hydrogen ions) are gained or lost because transfers of hydrogen atoms involve transfers of electrons (H = H+ + e–). Thus, when a molecule loses hydrogen atoms, it becomes oxidized. Oxidation and reduction always occur together: As one material is oxidized, the electrons it loses are transferred to another material, reducing that material. In a redox reaction, we call the reactant that becomes reduced an oxidizing agent and the one that becomes oxidized a reducing agent. In both the combustion and the metabolism of glucose, glucose is the reducing agent and oxygen gas is the oxidizing agent. In a redox reaction, energy is transferred. Much of the energy originally present in the reducing agent becomes associated with the reduced product. (The rest remains in the reducing agent or is lost.) As we will see, some of the key reactions of glycolysis and cellular respiration are highly exergonic redox reactions.

The coenzyme NAD is a key electron carrier in redox reactions

In preceding Chapter, we described the role of coenzymes, small molecules that assist in enzyme-catalyzed reactions.

ADP acts as a coenzyme when it picks up energy released in an exergonic reaction and uses it to make ATP (an endergonic reaction). In a similar fashion, the coenzyme NAD (nicotinamide adenine dinucleotide) acts as an energy carrier, in this case in redox reactions.

NAD exists in two chemically distinct forms, one oxidized (NAD+) and the other reduced (NADH + H+). Both forms participate in biological redox reactions. The reduction reaction NAD+ + 2 H NADH + H+ is formally equivalent to the transfer of two hydrogen atoms (2 H+ + 2 e–). However, what is actually transferred is a hydride ion (H–, a proton and two electrons), leaving a free proton (H+). This notation emphasizes that reduction is accomplished by the addition of electrons. Oxygen is highly electronegative and readily accepts electrons from NADH. The oxidation of NADH + H+ by O₂, NADH + H+ + 1⁄2 O₂ NAD+ + H₂O is highly exergonic, with a ∆G = –52.4 kcal/mol (–219 kJ/mol). Note that the oxidizing agent appears here as “1⁄2 O₂” instead of “O.” This notation emphasizes that it is oxygen gas, O₂, that acts as the oxidizing agent. Just as ATP can be thought of as packaging free energy in bundles of about 12 kcal/mol (50 kJ/mol), NAD can be thought of as packaging free energy in bundles of approximately 50 kcal/mol (200 kJ/mol). NAD is the most common, but not the only, electron carrier in cells. As you will see, another carrier, FAD (flavin adenine dinucleotide), is also involved in transferring electrons during glucose metabolism.

An Overview: Releasing Energy from Glucose

Depending on the presence or absence of O₂, the energy-harvesting processes in cells use different combinations of metabolic pathways:

When O₂ is available as the final electron acceptor, four pathways operate. Glycolysis takes place first and is followed by three pathways of cellular respiration: pyruvate oxidation, the citric acid cycle and the respiratory chain (also known as the electron transport chain).

When O₂ is unavailable, pyruvate oxidation, the citric acid cycle, and the respiratory chain do not function, and the pyruvate produced by glycolysis is further metabolized by fermentation. These five metabolic pathways, which we will consider at a time, have different locations in the cell.

Glycolysis: From Glucose to Pyruvate

We begin our discussion of the energy-harvesting pathways with glycolysis which begins glucose metabolism. Glycolysis takes place in the cytoplasm of cells. It converts glucose to pyruvate, produces a small amount of energy and does not generate CO₂. In glycolysis, a reduced fuel molecule, glucose, gets partially oxidized and in the process releases some of its energy. After ten enzyme-catalyzed reactions, the end products of glycolysis are two molecules of pyruvate (pyruvic acid)*. These reactions are accompanied by the net formation of two molecules of ATP and by the reduction of two molecules of NAD+ to two molecules of NADH + H+ for each molecule of glucose. Glycolysis can be divided into two stages: energy-investing reactions that use ATP, and energy-harvesting reactions that produce ATP.

The energy-investing reactions of glycolysis require ATP

The first five reactions of glycolysis are endergonic; that is, the cell is investing free energy in the glucose molecule, rather than releasing energy from it. In two separate reactions, the energy of two molecules of ATP is invested in attaching two phosphate groups to the glucose molecule to form fructose 1,6-bisphosphate,* which has a free energy substantially higher than that of glucose. Later, these phosphate groups will be transferred to ADP to make new molecules of ATP. Although both of these first steps of glycolysis use ATP as one of their substrates, each is catalyzed by a different, specific enzyme. The enzyme hexokinase catalyzes reaction 1, in which a phosphate group from ATP is attached to the sixcarbon glucose molecule, forming glucose 6-phosphate. (A kinase is any enzyme that catalyzes the transfer of a phosphate group from ATP to another substrate.) In reaction 2, the six-membered glucose ring is rearranged into a fivemembered fructose ring. In reaction 3, the enzyme phosphofructokinase adds a second phosphate (taken from another ATP) to the fructose ring, forming a six-carbon sugar, fructose 1,6-bisphosphate. Reaction 4 opens up and cleaves the six-carbon sugar ring to give two different three-carbon sugar phosphates: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. In reaction 5, one of those products, dihydroxyacetone phosphate, is converted into a second molecule of the other one, glyceraldehyde 3-phosphate (G3P). By this time—the halfway point of the glycolytic pathway— the following things have happened:

Two molecules of ATP have been invested.

The six-carbon glucose molecule has been converted into two molecules of a three-carbon sugar phosphate, glyceraldehydes 3-phosphate (G3P, a triose phosphate).

The energy-harvesting reactions of glycolysis yield NADH + H+ and ATP

With the investment of two ATPs, the first five reactions of glycolysis have rearranged the six-carbon sugar glucose and split it into two three-carbon sugar phosphates (G3P). In the discussion that follows, remember that each reaction occurs twice for each glucose molecule going through glycolysis because each glucose molecule has been split into two molecules of G3P. It is the fate of G3P that now concerns us—its transformation will generate both NADH + H+ and ATP.

Producing NADH + H+.

Reaction 6 is catalyzed by the enzyme triose phosphate dehydrogenase, and its end product is a phosphate ester, 1,3-bisphosphoglycerate (BPG). Reaction 6 is an oxidation and it is accompanied by an enormous drop in free energy — more than 100 kcal of energy per mole of glucose is released in this extremely exergonic reaction. If this big energy drop were simply a loss of heat, glycolysis would not provide useful energy to the cell. However, rather than being lost as heat, this energy is stored as chemical energy by reducing two molecules of NAD+ to make two molecules of NADH + H+. Because NAD+ is present in small amounts in the cell, it must be recycled to allow glycolysis to continue; if none of the NADH is oxidized back to NAD+, glycolysis comes to a halt. The metabolic pathways that follow glycolysis carry out this oxidation, as we will see.

Producing ATP.

In reactions 7–10, the two phosphate groups of BPG are transferred one at a time to molecules of ADP, with a rearrangement in between. More than 20 kcal (83.6 kJ/mol) of free energy is stored in ATP for every mole of BPG broken down. Finally, we are left with two moles of pyruvate for every mole of glucose that entered glycolysis.

The enzyme-catalyzed transfer of phosphate groups from donor molecules to ADP molecules (as in reaction 7) is called substrate-level phosphorylation. (Phosphorylation is the addition of a phosphate group to a molecule. Substrate-level phosphorylation is distinguished from the oxidative phosphorylation carried out by the respiratory chain, which we will discuss later in the chapter.) As an example of substrate-level phosphorylation, when G3P reacts with a phosphate group (Pi) and NAD+ in reaction 6, a second phosphate is added, an aldehyde is oxidized to a carboxylic acid, NAD+ is reduced and BPG is formed. The oxidation provides so much energy that the newly added phosphate group is linked to the rest of the molecule by a covalent bond that has even more energy than the terminal phosphate-to-phosphate bond of ATP. Another example of substrate-level phosphorylation occurs in reaction 7, where phosphoglycerate kinase catalyzes the transfer of a phosphate group from BPG to ADP forming ATP. Both reactions 6 and 7 are exergonic, even though a substantial amount of energy is consumed in the formation of ATP. A review of the glycolytic pathway shows that at the beginning of glycolysis, two molecules of ATP are used per molecule of glucose, but that ultimately four molecules of ATP are produced (two for each of the two BPG molecules)— a net gain of two ATP molecules and two NADH + H+.

Glycolysis is followed by cellular respiration (if O₂ is present) or fermentation (if no O₂ is present). The first reaction of cellular respiration is the oxidation of pyruvate.

Pyruvate Oxidation

The oxidation of pyruvate to acetate and its subsequent conversion to acetyl CoA is the link between glycolysis and all the other reactions of cellular respiration.

Coenzyme A (CoA), which is attached to the acetyl group to form acetyl CoA, is a complex molecule composed of a nucleotide, the vitamin pantothenic acid and a sulfur-containing group that is responsible for binding the two-carbon acetate molecule. Acetyl CoA formation is a multi-step reaction catalyzed by the pyruvate dehydrogenase complex, an enormous enzyme complex that is attached to the inner mitochondrial membrane. Pyruvate diffuses into the mitochondrion where a series of coupled reactions takes place:

1. Pyruvate is oxidized to a two-carbon acetyl group, and CO₂ is released.

2. Part of the energy from the oxidation is captured by the reduction of NAD+ to NADH + H+.

3. Some of the remaining energy is stored temporarily by the combining of the acetyl group with CoA, forming acetyl CoA: pyruvate + NAD+ + CoAAcetyl CoA + NADH + H+ + CO₂ Acetyl CoA has 7.5 kcal/mol (31.4 kJ/mol) more energy than simple acetate. Acetyl CoA can donate the acetyl group to acceptor molecules, much as ATP can donate phosphate groups to various acceptors. In the next section, we will see that the acetyl CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate.

The Citric Acid Cycle

Acetyl CoA is the starting point for the citric acid cycle (also called the Krebs cycle or the tricarboxylic acid cycle). This pathway of eight reactions completely oxidizes the two-carbon acetyl group to two molecules of carbon dioxide. The free energy released from these reactions is captured by ADP and the electron carriers NAD and FAD.

The metabolism of glucose to pyruvate is accompanied by a total drop in free energy of about 140 kcal/mol (585 kJ/mol). About a third of this energy is captured in the formation of ATP and reduced NAD (NADH + H+). Oxidizing pyruvate to acetate yields much additional free energy. Then, the citric acid cycle takes the acetyl group and essentially breaks it down to two molecules of CO₂, using the hydrogen atoms to reduce electron carriers and passing chemical free energy to those carriers in the process. The reduced carriers are oxidized in the respiratory chain which transfers an enormous amount of free energy to ATP.

The inputs to the citric acid cycle are acetate (in the form of acetyl CoA), water, and oxidized electron carriers (NAD+ and FAD).

The outputs are carbon dioxide, reduced electron carriers (NADH + H+ and FADH2), and a small amount of ATP.

Overall, for each acetyl group, the citric acid cycle removes two carbons as CO₂ and uses four pairs of hydrogen atoms to reduce electron carriers.

The citric acid cycle produces two CO₂ molecules and reduced carriers

Acetyl CoA enters the citric acid cycle from pyruvate oxidation, which has released CO₂. At the beginning of the citric acid cycle, acetyl CoA, which has two carbon atoms in its acetyl group, reacts with a four-carbon acid, oxaloacetate, to form the six-carbon compound citrate (citric acid). The remainder of the cycle consists of a series of enzyme-catalyzed reactions in which citrate is converted to a new four-carbon molecule of oxaloacetate. This new oxaloacetate can react with a second acetyl CoA, producing a second molecule of citrate and thus enabling the cycle to continue. The citric acid cycle is maintained in a steady state — that is, although the intermediate compounds in the cycle enter and leave, the concentrations of those intermediates do not change much. Also, recall that energy is released upon oxidation and stored in either ATP, FADH2 or NADH + H+. The energy temporarily stored in acetyl CoA drives the formation of citrate from oxaloacetate (reaction 1). During this reaction, the coenzyme Amolecule is removed and can be reused. In reaction 2, the citrate molecule is rearranged to form isocitrate. In reaction 3, a CO₂ molecule and two hydrogen atoms are removed, converting isocitrate to ketoglutarate. This reaction produces a large drop in free energy, some of which is stored in NADH + H+. Like the oxidation of pyruvate to acetyl CoA, reaction 4 of the citric acid cycle is complex. The five-carbon -ketoglutarate molecule is oxidized to the four-carbon molecule succinate. In the process, CO₂ is given off, some of the oxidation energy is stored in NADH + H+, and some of the energy is preserved temporarily by combining succinate with CoA to form succinyl CoA. In reaction 5, some of the energy in succinyl CoA is harvested to make GTP (guanosine triphosphate) from GDP and Pi, which is another example of substrate-level phosphorylation. GTP is then used to make ATP from ADP. Free energy is released in reaction 6, in which the succinate released from succinyl CoA in reaction 5 is oxidized to fumarate. In the process, two hydrogens are transferred to an enzyme that contains the carrier FAD. After a molecular rearrangement (reaction 7), one more NAD+ reduction occurs, producing oxaloacetate from malate (reaction 8). These two reactions illustrate a common biochemical mechanism: Water (H2O) is added in reaction 7 to form an —OH group, and then the H from that —OH group is removed in reaction 8 to reduce NAD+ to NADH + H+. The final product, oxaloacetate, is ready to combine with another acetyl group from acetyl CoA and go around the cycle again. The citric acid cycle operates twice for each glucose molecule that enters glycolysis (once for each pyruvate that enters the mitochondrion). Although most of the enzymes of the citric acid cycle are located in the mitochondrial matrix, there are two exceptions: succinate dehydrogenase, which catalyzes reaction 6 and ketoglutarate dehydrogenase, which catalyzes reaction 4.

These enzymes are integral membrane proteins of the inner mitochondrial membrane. Generations of students have asked the question, “Why did this complicated system evolve to achieve the simple goal of oxidizing two acetyl groups to two molecules of CO2?” There are three reasons:

First, the cycle includes molecules that have other roles in the cell. As we will see later in this chapter, the intermediates of the citric acid cycle are themselves catabolic (breakdown) products or anabolic (synthesis) building blocks of other molecules, such as amino acids andnucleotides.

Second, the citric acid cycle is far more efficient at harvesting energy from acetyl CoA than any single reaction could be.

Third, evolution is a conservative, add-on process. It rarely operates by inventing an entirely new process.

The Respiratory Chain: Electrons, Protons, and ATP Production

Pyruvate oxidation and the operation of the citric acid cycle generate large amounts of reduced electron carriers containing trapped energy. To liberate this energy and produce ATP, something must happen to these reduced carriers. Furthermore, without NAD+ and FAD, the oxidative steps of glycolysis, pyruvate oxidation, and the citric acid cycle could not occur. To regenerate NAD+ and FAD, the reduced forms of these carriers must have some way to get rid of their hydrogens (H+ + e–). The fate of these protons and electrons is the rest of the story of cellular respiration. The story hasthree parts:

1. The electrons pass through a series of membrane-associated electron carriers called the respiratory chain or the electron transport chain.

2. The flow of electrons along the chain accomplishes the active transport of protons across the inner mitochondrial membrane, out of the matrix, creating a proton concentration gradient.

3. The protons diffuse back into the mitochondrial matrix through a proton channel, which couples this diffusion to the synthesis of ATP.

The overall process of ATP synthesis resulting from electron transport through the chain is called oxidative phosphorylation.

Before we proceed with the details of oxidative phosphorylation, let’s reflect on an important question: Why should the respiratory chain have so many components and complex processes? Why, for example, don’t cells use the following single step?

NADH + H+ + 1⁄2 O2 NAD+ + H2O

Fundamentally, this would be an untamable reaction. It would be very exergonic—rather like setting off a stick of dynamite in the cell. There is no biochemical way to harvest that burst of energy efficiently and put it to physiological use (that is, no metabolic reaction that is so endergonic as to consume a significant fraction of that energy in a single step). To control the release of energy during the oxidation of glucose in a cell, evolution has produced the lengthy electron transport chain we observe today: a series of reactions, each releasing a small, manageable amount of energy.

The respiratory chain transports electrons and releases energy

The respiratory chain contains large integral proteins, smaller mobile proteins, and even a smaller lipid molecule:

Four large protein complexes containing carrier molecules and their associated enzymes are integral proteins of the inner mitochondrial membrane in eukaryotes.

Cytochrome c is a small peripheral protein that lies in the space between the inner and outer mitochondrial membranes. It is loosely attached to the inner membrane.

A nonprotein component called ubiquinone (abbreviated Q) is a small, nonpolar molecule that moves freely within the hydrophobic interior of the phospholipids bilayer of the inner membrane.

NADH + H+ passes electrons to Q by way of the first large protein complex, called NADH-Q reductase, which contains twenty-six polypeptides and attached prosthetic groups. NADH-Q reductase passes the electrons to Q, forming QH₂. The second complex, succinate dehydrogenase, passes electrons to Q from FADH₂ during the formation of fumarate from succinate in reaction 6 of the citric acid cycle. These electrons enter the chain later than those from NADH. The third complex, cytochrome c reductase, with ten subunits, receives electrons from QH₂ and passes them to cytochrome c. The fourth complex, cytochrome c oxidase, with eight subunits, receives electrons from cytochrome c and passes them to oxygen, which with these extra electrons (1⁄2 O₂ –) picks up two hydrogen ions (H+) to form H₂O. The electron carriers of the respiratory chain (including those contained in the three protein complexes) differ as to how they change when they become reduced. NAD+, for example, accepts H– (a hydride ion — one proton and two electrons), leaving the proton from the other hydrogen atom to float free: NADH + H+. Other carriers, including Q, bind both protons and both electrons becoming, for example, QH2. The remainder of the chain, however, is only an electron transport process. Electrons, but not protons, are passed from Q to cytochrome c. An electron from QH₂ reduces a cyto-chrome’s Fe3+ to Fe2+. The fate of the protons will be discussed below. Electron transport within each of the three protein complexes results, as we’ll see, in the pumping of protons across the inner mitochondrial membrane, and the return of the protons across the membrane is coupled to the formation of ATP. Thus the energy originally contained in glucose and other foods is finally captured in the cellular energy currency, ATP. For each pair of electrons passed along the chain from NADH + H+ to oxygen, three molecules of ATP are formed. If only electrons are carried through the final reactions of the respiratory chain, what happens to the protons? How are proton movements coupled to the production of ATP?

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.

Regulating Energy Pathways

We have described the relationships between metabolic pathways and noted that these pathways work together to provide homeostasis in the cell and organism. But how does the cell regulate interconversions between these pathways to maintain constant metabolic pools? Consider what happens to the starch in your burger bun. In the digestive system, starch is hydrolyzed to glucose which enters the blood for distribution to the rest of the body. Before this happens, however, a “decision” must be made: Is there already enough glucose in the blood to supply the body’s needs? If there is, the excess glucose is converted to stored glycogen in the liver. If not enough glucose is supplied by food, liver glycogen is broken down or other molecules are used to make glucose by gluconeogenesis. The end result is that the level of glucose in the blood is remarkably constant. For now, it is important to realize that the interconversions of glucose involve many steps, each catalyzed by an enzyme, and it is here that controls often reside. Glycolysis, the citric acid cycle, and the respiratory chain are regulated by allosteric control of the enzymes involved. These negative and positive feedback control mechanisms are used at many points in the energyharvesting pathways, which are summarized in Figure . The main control point in glycolysis is the enzyme phosphofructokinase (reaction 3). This enzyme is allosterically inhibited by ATP and activated by ADP or AMP. As long as fermentation proceeds, yielding a relatively small amount of ATP, phosphofructokinase operates at full efficiency. But when cellular respiration begins producing 18 times more ATP than fermentation does, the abundant ATP allosterically inhibits the enzyme, and the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate declines, as does the rate of glucose utilization.

The main control point in the citric acid cycle is the enzyme isocitrate dehydrogenase, which converts isocitrate to ketoglutarate (reaction 3). NADH + H+ and ATP are feedback inhibitors of this reaction; ADP and NAD+ are activators. If too much ATP is accumulating, or if NADH + H+ is being produced faster than it can be used by the respiratory chain, the conversion of isocitrate is slowed, and the citric acid cycle is essentially shut down. A shutdown of the citric acid cycle would cause large amounts of isocitrate and citrate to accumulate if the conversion of acetyl CoA to citrate were not also slowed by abundant ATP and NADH + H+. However, a certain excess of citrate does accumulate, and this excess acts as an additional negative feedback inhibitor to slow the fructose 6-phosphate reaction early in glycolysis. Consequently, if the citric acid cycle has been slowed down because of abundant ATP (and not because of a lack of oxygen), glycolysis is shut down as well. Both processes resume when the ATP level falls and they are needed again. Allosteric control keeps these processes in balance. Another control point involves a method for storing excess acetyl CoA. If too much ATP is being made and the citric acid cycle shuts down, the accumulation of citrate switches acetyl CoA to the synthesis of fatty acids for storage. This is one reason why people who eat too much accumulate fat. These fatty acids may be metabolized later to produce more acetyl CoA.