OxfordAQA International AS and A-level Biology: Respiration, Krebs cycle and RQ (9610)

This study guide is aligned to the OxfordAQA International AS and A-level Biology (9610) specification, Version 5.1 (International AS exams May/June 2017 onwards, International A-level exams May/June 2018 onwards). It covers section 3.3.3, Respiration, in four parts: 3.3.3.1 glycolysis, 3.3.3.2 anaerobic respiration, 3.3.3.3 aerobic respiration and 3.3.3.4 respiratory substrates. It also covers required practical 8. Section 3.3.3 sits in Unit 3, Populations and genes, which the specification lists as International A-level only.

Use it with the revision notes and the practice questions. The course hub is OxfordAQA A-level Biology, the printable checklist tracks every statement, and the free diagnostics show where to start.

What this unit covers

Section	What you must be able to do
3.3.3.1	Describe glycolysis in the cytoplasm: phosphorylation of glucose using ATP, then oxidation of triose phosphate to pyruvate with a net gain of ATP and reduced NAD
3.3.3.2	Explain how pyruvate is converted to ethanol and carbon dioxide, or to lactate, using reduced NAD, and why this regenerates NAD
3.3.3.3	Describe pyruvate entry to the matrix, the link reaction, acetyl coenzyme A, the Krebs cycle and oxidative phosphorylation with ATP synthase
3.3.3.4	Explain how lipids and amino acids are respired, calculate RQ, and judge what an RQ does and does not show
Required practical 8	Investigate a variable such as substrate or temperature on the rate of respiration of an organism such as yeast or a locust

3.3.3.1 Glycolysis

Glycolysis happens in the cytoplasm. It is common to aerobic and anaerobic respiration, so every respiring cell in this unit starts here. It needs no oxygen.

1. Phosphorylation. Glucose is phosphorylated to glucose phosphate using ATP. A second phosphate from a second ATP gives a six-carbon sugar bisphosphate. Phosphorylation makes the sugar more reactive.
2. Splitting. The six-carbon molecule splits into two molecules of triose phosphate (three carbons each).
3. Oxidation. Each triose phosphate is oxidised to pyruvate. Hydrogen is removed and taken up by NAD, which becomes reduced NAD.
4. ATP formation. Phosphate groups are transferred from intermediates to ADP. This is substrate-level phosphorylation. Four ATP are made per glucose.

Net result for one glucose: 2 ATP used, 4 ATP made, so a net gain of 2 ATP, 2 reduced NAD and 2 pyruvate.

Worked example 1. A cell extract oxidises 0.15 mol of glucose by glycolysis. Calculate the ATP used, ATP made, net ATP and reduced NAD.

• ATP used = 2 × 0.15 = 0.30 mol.
• ATP made = 4 × 0.15 = 0.60 mol.
• Net ATP = 0.60 - 0.30 = 0.30 mol.
• Reduced NAD = 2 × 0.15 = 0.30 mol, and pyruvate = 0.30 mol.

3.3.3.2 Anaerobic respiration

Without oxygen the later stages cannot run, so reduced NAD would build up and glycolysis would stop for lack of NAD. Anaerobic respiration solves this. Pyruvate is converted using reduced NAD, which is oxidised back to NAD. The NAD can then be used in further glycolysis, so a small amount of ATP keeps being made.

	Lactate pathway	Ethanol pathway
Found in	Muscle and other animal cells	Yeast and some plant cells
Change	Pyruvate + reduced NAD gives lactate + NAD	Pyruvate gives ethanal and carbon dioxide; ethanal + reduced NAD gives ethanol + NAD
Carbon dioxide released?	No	Yes
Fate of product	Lactate can be turned back into pyruvate once oxygen is available	Ethanol is a waste product and cannot be reversed

Neither pathway makes extra ATP. The only ATP is the net 2 from glycolysis. The pathway does not itself make ATP; its job is to regenerate NAD.

3.3.3.3 Aerobic respiration

Pyruvate enters the mitochondrial matrix by active transport. Everything below happens inside the mitochondrion.

The link reaction (matrix)

Pyruvate is oxidised to acetate, producing reduced NAD. Carbon dioxide is also lost (decarboxylation). Acetate combines with coenzyme A to form acetyl coenzyme A (acetyl CoA, two carbons). One link reaction gives one carbon dioxide, one reduced NAD and one acetyl CoA, and it happens twice per glucose.

The Krebs cycle (matrix)

Acetyl CoA reacts with a four-carbon molecule. Coenzyme A is released, ready to be used again, and a six-carbon molecule is produced. This enters the Krebs cycle, a series of oxidation-reduction reactions. Carbon dioxide is released twice and hydrogen is removed several times, so the four-carbon molecule is regenerated.

Per acetyl CoA the cycle generates:

Product	Number
Carbon dioxide	2
Reduced NAD	3
Reduced FAD	1
ATP (substrate-level)	1

Reduced NAD and reduced FAD are the reduced coenzymes. Most of the energy from glucose is now held in them.

Worked example 2. A mitochondrial preparation is given 0.40 mmol of pyruvate. Calculate the carbon dioxide, reduced NAD, reduced FAD and ATP made by the link reaction and Krebs cycle together. Assume oxidative phosphorylation yields 2.5 ATP for every reduced NAD and 1.5 for every reduced FAD (data supplied). Find the ATP from the reduced coenzymes.

• Per pyruvate: carbon dioxide 1 + 2 = 3; reduced NAD 1 + 3 = 4; reduced FAD 1; ATP 1.
• Carbon dioxide = 3 × 0.40 = 1.2 mmol.
• Reduced NAD = 4 × 0.40 = 1.6 mmol; reduced FAD = 0.40 mmol; ATP (Krebs) = 0.40 mmol.
• ATP from reduced coenzymes = (1.6 × 2.5) + (0.40 × 1.5) = 4.0 + 0.60 = 4.6 mmol.

Oxidative phosphorylation (inner mitochondrial membrane)

1. Reduced NAD and reduced FAD give up electrons (with protons) to the electron transfer chain in the inner membrane. NAD and FAD are re-formed and return to earlier stages.
2. Electrons pass down the chain through a series of carriers. Energy released as they move is used to pump protons out of the matrix, across the inner membrane, so they collect between the two membranes.
3. This builds a proton gradient. The inner membrane is impermeable to protons except through channels.
4. Protons flow back into the matrix through ATP synthase, an enzyme embedded in the membrane. The flow drives synthesis of ATP from ADP and phosphate.
5. At the end of the chain, oxygen accepts the electrons and combines with protons to form water. Oxygen is the final electron acceptor.

Without oxygen the chain stops, the coenzymes stay reduced, and the Krebs cycle and link reaction stop for lack of NAD and FAD. This is why aerobic respiration cannot continue without oxygen. The folded inner membrane (cristae) gives a larger area for chains and ATP synthase.

Worked example 3. Use the data supplied (2.5 ATP per reduced NAD, 1.5 per reduced FAD, and reduced NAD from glycolysis treated the same) to find the ATP from one glucose respired aerobically.

• Glycolysis: net 2 ATP + 2 reduced NAD (5 ATP) = 7.
• Link reaction: 2 reduced NAD = 5.
• Krebs cycle: 2 ATP + 6 reduced NAD (15) + 2 reduced FAD (3) = 20.
• Total = 7 + 5 + 20 = 32 ATP, compared with 2 anaerobically, so 16 times as much (the anaerobic yield is 6.25% of the aerobic one).

Yields differ between textbooks. Use the figures a question gives you.

3.3.3.4 Respiratory substrates

Carbohydrate is not the only fuel. Digestion products of fat and protein are also respired, and they join the pathway at the Krebs cycle or earlier.

• Lipids are hydrolysed to glycerol and fatty acids. Glycerol enters at glycolysis, as triose phosphate. Fatty acid chains are cut into two-carbon pieces, each forming acetyl CoA. Fatty acids contain many hydrogen atoms, so they give many reduced coenzymes per gram. This is why lipids store more energy per gram than carbohydrate.
• Amino acids have their amino group removed (deamination). The remaining carbon skeleton enters respiration, for example as pyruvate, acetyl CoA or a Krebs cycle intermediate. The nitrogen is excreted, for example as urea.

Respiratory quotient

RQ = carbon dioxide produced ÷ oxygen consumed. The two gases must be in the same units, in quantities proportional to the number of molecules: moles or volumes are both acceptable. RQ has no units.

Substrate	Typical RQ
Lipid	close to 0.7
Protein	close to 0.9
Carbohydrate	close to 1.0

Worked example 4: RQ from an equation. Oleic acid is respired: C18H34O2 + 25.5 O2 → 18 CO2 + 18 H2O. RQ = 18 ÷ 25.5 = 0.71, so the substrate is a lipid. For glucose, C6H12O6 + 6 O2 → 6 CO2 + 6 H2O, RQ = 6 ÷ 6 = 1.0. Lipids have fewer oxygen atoms in the molecule, so more oxygen is needed to oxidise them.

Worked example 5: RQ from a respirometer. Mealworms are placed in a respirometer. The capillary has a diameter of 1.0 mm. With potassium hydroxide solution (which absorbs carbon dioxide) the drop moves 45 mm in 10 minutes. In a second tube without it, the drop moves 9 mm.

• Oxygen used = π × 0.50² × 45 = 35.3 mm³.
• Without absorber, the net change is oxygen taken in minus carbon dioxide released = π × 0.50² × 9 = 7.07 mm³.
• Carbon dioxide = 35.3 - 7.07 = 28.3 mm³.
• RQ = 28.3 ÷ 35.3 = 0.80.

The radius cancels, so (45 - 9) ÷ 45 gives the same answer.

Why conclusions are tentative

A value between 0.7 and 1.0 points to a mixed diet of fuels. Be ready to comment on these limits:

• Several substrates are usually respired at once, so one value can fit more than one mixture.
• Some tissues also make carbon dioxide or use oxygen in other processes, and plants photosynthesise in light.
• Anaerobic respiration in yeast releases carbon dioxide without using oxygen, so the RQ rises above 1. Lactate fermentation releases none, so the RQ falls.
• Making fat from carbohydrate can release carbon dioxide with little oxygen use, which raises the measured RQ above 1.
• Measurements may be affected by temperature changes, leaks, and different solubilities of the gases.

Required practical 8: respiration rate of yeast

The practical investigates a variable such as substrate or temperature on the rate of respiration of an organism such as yeast or a locust. The specification says the International A2 written papers test knowledge of the procedures and evaluation of the techniques, and may ask you to interpret specimen results.

• Measuring rate: carbon dioxide collected in a gas syringe or over water, oxygen uptake in a respirometer with carbon dioxide absorbed, or time for a redox indicator such as methylene blue to lose its colour.
• Controls: use boiled, killed yeast as a control, keep the volume and concentration of the culture the same, and use a water bath for temperature.
• Repeats: at least three at each value, then a mean.

Worked example 6. Yeast in 5% glucose solution released this volume of carbon dioxide in 10 minutes.

Temperature / °C	10	22	34	46	58
Volume / cm³	1.4	4.8	10.2	6.5	0.3

Rates in cm³ per min are 0.14, 0.48, 1.02, 0.65 and 0.03. The rate at 34 °C is 10.2 ÷ 4.8 = 2.1 times that at 22 °C, because molecules move faster and enzyme-substrate collisions become more frequent. Between 34 °C and 46 °C the volume falls by (6.5 - 10.2) ÷ 10.2 × 100 = -36%, which suggests enzymes are denaturing. The optimum lies somewhere between 22 °C and 46 °C, so more temperatures between them are needed.

Common errors

• Saying glycolysis makes 2 ATP. It makes 4 and uses 2.
• Writing that the Krebs cycle “makes lots of ATP”. Most ATP comes from oxidative phosphorylation.
• Placing oxygen in the Krebs cycle. It takes part only where electrons leave the electron transfer chain.
• Writing that anaerobic respiration “makes ATP from pyruvate”. Its role is to regenerate NAD.
• Dividing oxygen by carbon dioxide when calculating RQ.

Test yourself with the practice questions and revise from the revision notes.

Official syllabus

OxfordAQA International AS and A-level Biology (9610), Version 5.1, section 3.3.3 (Respiration), for International AS exams May/June 2017 onwards and International A-level exams May/June 2018 onwards. Published by OxfordAQA.