What you'll learn
Respiration: glycolysis and the Krebs cycle covers the first two stages by which organisms release the energy stored in organic molecules. At CAPE level you must know these pathways by stage, location, input and output, and be able to account for every carbon atom and every reduced coenzyme. The reason for that detail is that the reduced coenzymes produced here are what drive ATP synthesis in the final stage, so the yields determined in glycolysis and the Krebs cycle determine the yield of the whole process. By the end of this topic you should be able to describe glycolysis and the link reaction and the Krebs cycle in full, state the location and net yield of each, explain anaerobic respiration in animals and in yeast, compare respiratory substrates, and describe how respiration rate is measured.
Key terms and definitions
Respiration — the enzyme-controlled release of energy from organic molecules, producing ATP
ATP — adenosine triphosphate, the universal immediate energy currency of cells
Glycolysis — the splitting of glucose into two molecules of pyruvate in the cytoplasm
Phosphorylation — the addition of a phosphate group to a molecule
Substrate-level phosphorylation — the direct formation of ATP by transfer of a phosphate group from a substrate
Link reaction — the conversion of pyruvate to acetyl coenzyme A in the mitochondrial matrix
Decarboxylation — the removal of a carbon dioxide molecule
Dehydrogenation — the removal of hydrogen, which is accepted by a coenzyme
Krebs cycle — the cyclical series of reactions in the matrix that completes the oxidation of acetyl groups
NAD and FAD — coenzymes that accept hydrogen and carry it to the electron transport chain
Anaerobic respiration — the release of energy from organic molecules without oxygen
Respiratory quotient — the ratio of carbon dioxide produced to oxygen consumed, abbreviated RQ
Core concepts
ATP as the energy currency
ATP consists of adenine, ribose and three phosphate groups. Hydrolysis of the terminal phosphate releases a relatively small, usable quantity of energy and produces ADP and inorganic phosphate, a reaction catalysed by ATP hydrolase.
ATP is described as the universal energy currency because it is used by all organisms and in all energy-requiring processes: active transport, muscle contraction, protein synthesis, nerve impulse transmission and the anabolic reactions of metabolism.
Its suitability rests on several properties. It releases energy in small, manageable quantities, so little is wasted as heat. It is readily hydrolysed in a single step, so energy is available immediately. It is soluble and moves easily within the cell. And it is easily regenerated from ADP and phosphate, so the same molecules are recycled continuously rather than stored in quantity.
ATP is not a long-term energy store; cells hold only a small quantity and turn it over rapidly.
Glycolysis
Glycolysis occurs in the cytoplasm and does not require oxygen. It is common to both aerobic and anaerobic respiration.
The pathway has two phases. In the phosphorylation phase, glucose, a six-carbon sugar, is phosphorylated using two molecules of ATP, forming hexose bisphosphate. This investment of ATP makes the molecule more reactive and less likely to leave the cell.
Hexose bisphosphate then splits into two molecules of triose phosphate, each of three carbons.
In the oxidation phase, each triose phosphate is oxidised by dehydrogenation to pyruvate. The hydrogen removed is accepted by NAD, forming reduced NAD, and two molecules of ATP are produced from each triose phosphate by substrate-level phosphorylation.
The accounting is examinable. Two ATP are used and four are produced, so the net gain is two ATP. Two molecules of reduced NAD are produced, and two molecules of pyruvate.
The link reaction
Pyruvate produced in the cytoplasm is actively transported into the mitochondrial matrix, where the link reaction occurs. Oxygen is required for this and all subsequent stages.
Each pyruvate, with three carbons, undergoes decarboxylation, losing one carbon as carbon dioxide, and dehydrogenation, with the hydrogen accepted by NAD to form reduced NAD.
The resulting two-carbon acetyl group combines with coenzyme A to form acetyl coenzyme A, which carries it into the Krebs cycle.
Per glucose molecule, since there are two pyruvate molecules, the link reaction produces two carbon dioxide, two reduced NAD and two acetyl coenzyme A. No ATP is produced directly.
The Krebs cycle
The Krebs cycle occurs in the mitochondrial matrix. Each turn processes one acetyl group, so there are two turns per glucose molecule.
Acetyl coenzyme A delivers its two-carbon acetyl group to a four-carbon compound, oxaloacetate, forming a six-carbon compound, citrate. Coenzyme A is released and returns to the link reaction.
Citrate then undergoes a series of reactions involving decarboxylation and dehydrogenation. Two molecules of carbon dioxide are released per turn, reducing the compound back to the four-carbon oxaloacetate, which is regenerated and can accept another acetyl group.
Per turn, the cycle produces three molecules of reduced NAD, one of reduced FAD, one of ATP by substrate-level phosphorylation, and two of carbon dioxide.
Per glucose molecule, doubling these gives six reduced NAD, two reduced FAD, two ATP and four carbon dioxide.
The essential point about the Krebs cycle, and the one questions test, is that its purpose is not to make ATP directly — it makes only two per glucose — but to generate large numbers of reduced coenzymes. These carry hydrogen and electrons to the electron transport chain, where the great majority of the ATP is synthesised.
Totals from the first three stages
Combining the three stages per glucose molecule: glycolysis gives a net two ATP, two reduced NAD and two pyruvate; the link reaction gives two reduced NAD and two carbon dioxide; and the Krebs cycle gives two ATP, six reduced NAD, two reduced FAD and four carbon dioxide.
The running totals are therefore four ATP by substrate-level phosphorylation, ten reduced NAD, two reduced FAD and six carbon dioxide — which accounts for all six carbon atoms of the original glucose molecule.
Being able to state that all the carbon dioxide of aerobic respiration comes from the link reaction and the Krebs cycle, and none from glycolysis, is a standard examination point.
Anaerobic respiration
In the absence of oxygen, the electron transport chain cannot operate because there is no final electron acceptor. Reduced NAD therefore accumulates, and since NAD is present in limited quantity, glycolysis would stop once it was all reduced.
Anaerobic pathways exist to regenerate NAD so that glycolysis can continue, and this is their sole purpose.
In animals, pyruvate is reduced to lactate. The hydrogen comes from reduced NAD, which is thereby oxidised back to NAD and can return to glycolysis. The lactate accumulates in muscle and is later transported in the blood to the liver, where it is converted back to pyruvate or to glucose when oxygen is available. The oxygen required to do this is the oxygen debt.
In yeast and plants, pyruvate is first decarboxylated to ethanal, releasing carbon dioxide, and the ethanal is then reduced to ethanol, again oxidising reduced NAD back to NAD. This is alcoholic fermentation, used in brewing for the ethanol and in bread making for the carbon dioxide.
The yield of anaerobic respiration is only the net two ATP from glycolysis, compared with around thirty to thirty-two for aerobic respiration, because the glucose is only partially broken down and the reduced coenzymes are not used to generate ATP.
Respiratory substrates and RQ
Carbohydrates are the usual substrate, entering as glucose at the start of glycolysis.
Lipids are hydrolysed to glycerol and fatty acids. Glycerol is converted to triose phosphate and enters glycolysis. Fatty acids are broken down into two-carbon fragments that enter the Krebs cycle as acetyl coenzyme A. Because fatty acids contain many hydrogen atoms relative to oxygen, they yield a great many reduced coenzymes and therefore far more ATP per gram than carbohydrate.
Proteins are hydrolysed to amino acids, which are deaminated in the liver, with the amino group converted to urea. The remaining carbon skeleton enters the pathway as pyruvate or as a Krebs cycle intermediate depending on the amino acid.
The respiratory quotient is the volume of carbon dioxide produced divided by the volume of oxygen consumed. It is approximately 1.0 for carbohydrate, 0.7 for lipid and 0.9 for protein, so an RQ value indicates which substrate is being respired. A value above 1.0 indicates that some anaerobic respiration is occurring, since carbon dioxide is being produced without corresponding oxygen consumption.
Measuring respiration rate
A simple respirometer measures the rate of oxygen consumption by a small organism or by germinating seeds.
The organism is placed in a sealed tube containing soda lime or potassium hydroxide solution, which absorbs the carbon dioxide produced. As oxygen is consumed and the carbon dioxide is absorbed, the volume of gas in the tube decreases and a coloured liquid in an attached capillary tube moves towards the organism.
The distance moved in a measured time, multiplied by the cross-sectional area of the capillary, gives the volume of oxygen consumed per unit time.
A control tube containing an equal volume of inert material, such as glass beads, is set up alongside. This compensates for changes in the liquid's position caused by fluctuations in temperature or atmospheric pressure rather than by respiration. The whole apparatus is kept in a water bath at constant temperature for the same reason.
Removing the carbon dioxide absorbent and repeating allows the carbon dioxide produced to be determined, and hence the RQ.
Worked examples
Example 1: Accounting for the products (5 marks)
State the net yield of ATP, reduced NAD and reduced FAD from one molecule of glucose up to the end of the Krebs cycle, and state where the carbon dioxide originates.
Glycolysis uses two ATP and produces four, giving a net two ATP, together with two reduced NAD and two pyruvate. No carbon dioxide is produced.
The link reaction occurs twice, producing two reduced NAD and two carbon dioxide.
The Krebs cycle turns twice, producing two ATP, six reduced NAD, two reduced FAD and four carbon dioxide.
The totals are therefore four ATP, ten reduced NAD and two reduced FAD. All six molecules of carbon dioxide come from the link reaction and the Krebs cycle, and none from glycolysis.
Example 2: Explaining anaerobic respiration (5 marks)
Explain why lactate production allows muscle cells to continue producing ATP in the absence of oxygen.
Glycolysis requires NAD to accept hydrogen when triose phosphate is oxidised to pyruvate. The cell contains only a limited quantity of NAD.
Without oxygen the electron transport chain cannot operate, because oxygen is the final electron acceptor. Reduced NAD therefore cannot be reoxidised by that route and would rapidly all be converted to the reduced form, at which point glycolysis would stop and no ATP could be made at all.
Reducing pyruvate to lactate uses the hydrogen from reduced NAD, regenerating NAD. This NAD returns to glycolysis, which can therefore continue and keep producing a net two ATP per glucose by substrate-level phosphorylation.
The yield is far lower than in aerobic respiration because the glucose is only partly broken down and the reduced coenzymes are not used for ATP synthesis, but it allows ATP production to continue for a limited period.
Example 3: Interpreting an RQ value (4 marks)
A germinating seed gives an RQ of 0.7, which later rises to 1.0. Suggest an explanation.
An RQ of 0.7 indicates that lipid is the main respiratory substrate. Many seeds store lipid because it yields more energy per gram than carbohydrate, which is an advantage when the food store must be compact. Lipids contain relatively little oxygen, so proportionally more oxygen must be taken in to oxidise them, giving a low ratio of carbon dioxide produced to oxygen consumed.
The rise to 1.0 indicates a change to carbohydrate as the main substrate. This would occur once the seedling has emerged and begun photosynthesising, producing carbohydrate that can be respired directly, or once the stored lipid has been exhausted and converted to carbohydrate.
Common mistakes and how to avoid them
The most frequent error is stating that glycolysis produces four ATP. Four are produced but two are used, so the net yield is two, and questions almost always ask for the net figure.
Students often place glycolysis in the mitochondrion. It occurs in the cytoplasm, which is why anaerobic organisms without mitochondria can still respire.
Another common slip is saying that the purpose of anaerobic respiration is to produce lactate or ethanol. The purpose is to regenerate NAD so that glycolysis can continue; the lactate and ethanol are by-products.
Many candidates state that carbon dioxide is produced in glycolysis. It is not; all of it comes from the link reaction and Krebs cycle.
In respirometer questions, answers frequently omit the control tube or fail to say what it compensates for. It corrects for changes due to temperature and pressure rather than respiration.
Finally, candidates often describe the Krebs cycle as the main source of ATP. It yields only two ATP per glucose directly; its importance is the reduced coenzymes it generates.
Exam technique for "Respiration: glycolysis and the Krebs cycle"
Learn each stage as a four-part entry: location, inputs, outputs and net ATP. Almost every question is answerable from that table.
Always state whether an ATP figure is gross or net, and give the net figure unless told otherwise.
When asked about anaerobic respiration, make the NAD regeneration the centre of the answer. It is the reason the pathway exists.
For RQ questions, quote the characteristic values and then interpret the specific figure given. A value above 1.0 always signals anaerobic respiration.
In respirometer questions, name the carbon dioxide absorbent, explain why the liquid moves, and describe the control. Three separate marks are usually available.
Quick revision summary
ATP is the universal energy currency, releasing energy in small usable amounts by a single hydrolysis and being readily regenerated. Glycolysis occurs in the cytoplasm without oxygen: glucose is phosphorylated using two ATP, split into two triose phosphate, and oxidised to two pyruvate, yielding four ATP gross and two net, plus two reduced NAD. The link reaction in the mitochondrial matrix decarboxylates and dehydrogenates each pyruvate to a two-carbon acetyl group joined to coenzyme A, giving two carbon dioxide and two reduced NAD per glucose. The Krebs cycle turns twice, each turn combining acetyl coenzyme A with four-carbon oxaloacetate to form six-carbon citrate, then releasing two carbon dioxide and producing three reduced NAD, one reduced FAD and one ATP, regenerating oxaloacetate. Totals are four ATP, ten reduced NAD, two reduced FAD and six carbon dioxide, all from the link reaction and Krebs cycle. Anaerobic respiration regenerates NAD by reducing pyruvate to lactate in animals or to ethanol and carbon dioxide in yeast, yielding only the two ATP from glycolysis. RQ is carbon dioxide produced divided by oxygen consumed, approximately 1.0 for carbohydrate, 0.7 for lipid and 0.9 for protein, and above 1.0 when anaerobic respiration occurs.