Respiration

In a nutshell

Every cell needs a constant supply of ATP, and respiration is how it makes it: the controlled, step-by-step oxidation of respiratory substrates such as glucose to release energy for ATP synthesis.

This subtopic covers the four stages of aerobic respiration (glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation), what anaerobic respiration does instead, and how the rate of respiration is measured.

Assumed knowledge: ATP, Cell structure (the mitochondrion), Transport across cell membranes.

Core content

The big picture: respiration produces ATP

Respiration is the release of energy from respiratory substrates to produce ATP, the cell's immediate energy currency.

Aerobic respiration happens in four stages, in two locations:

The first three stages mostly strip hydrogen atoms off the substrate and load them onto the coenzymes NAD and FAD (which become reduced NAD and reduced FAD). The final stage, oxidative phosphorylation, uses those reduced coenzymes to make the bulk of the ATP.

Stage 1: Glycolysis

Glycolysis is the first stage of both aerobic and anaerobic respiration. It happens in the cytoplasm and does not use oxygen, so it is an anaerobic process.

It splits one 6-carbon glucose molecule into two 3-carbon pyruvate molecules, in three spec steps:

1. Phosphorylation of glucose to glucose phosphate, using ATP. Two ATP molecules each transfer a phosphate to glucose, forming a phosphorylated 6-carbon sugar. This makes the sugar more reactive and is why the first steps cost ATP.
2. Production of triose phosphate. The phosphorylated 6-carbon sugar splits into two molecules of triose phosphate (3 carbons each).
3. Oxidation of triose phosphate to pyruvate, with a net gain of ATP and reduced NAD. Each triose phosphate is oxidised (hydrogen is removed) to pyruvate. The hydrogen reduces NAD to reduced NAD, and ATP is made directly by substrate-level phosphorylation.

Per glucose molecule, the tally is:

• 4 ATP made, 2 ATP used, so a net gain of 2 ATP
• 2 reduced NAD
• 2 pyruvate

Stage 2: The link reaction

If oxygen is available, each pyruvate is actively transported from the cytoplasm into the mitochondrial matrix (it cannot simply diffuse across).

In the matrix, each pyruvate is converted to acetylcoenzyme A:

• pyruvate is oxidised to acetate, and the hydrogen removed produces reduced NAD;
• a carbon is removed as carbon dioxide (decarboxylation);
• the 2-carbon acetate combines with coenzyme A to form acetylcoenzyme A.

Because glycolysis makes two pyruvate per glucose, the link reaction happens twice per glucose. So per glucose it yields 2 acetylcoenzyme A, 2 CO₂ and 2 reduced NAD. No ATP is made in the link reaction.

Stage 3: The Krebs cycle

The Krebs cycle is a closed cycle of oxidation-reduction reactions in the mitochondrial matrix. Like the link reaction, it turns twice per glucose.

Per turn of the cycle:

1. Acetylcoenzyme A delivers its 2-carbon acetate to a 4-carbon molecule, releasing coenzyme A and forming a 6-carbon molecule.
2. The 6-carbon molecule goes through a series of oxidation-reduction (redox) reactions that:
   • release 2 molecules of CO₂ (decarboxylation);
   • produce 3 reduced NAD and 1 reduced FAD;
   • make 1 ATP by substrate-level phosphorylation.
3. The 4-carbon molecule is regenerated, so the cycle can run again.

You do not need the names citrate and oxaloacetate for AQA; you can refer to the molecules by their number of carbons.

Stage 4: Oxidative phosphorylation

This is where most of the ATP is made, on the inner mitochondrial membrane. It uses the reduced NAD and reduced FAD collected in the first three stages.

The sequence:

1. Reduced NAD and reduced FAD are oxidised, releasing the hydrogen atoms they carried as protons (H⁺) and electrons.
2. The electrons pass along the electron transfer chain, a series of carriers, in a series of redox reactions. This releases energy.
3. The energy is used to pump protons from the matrix into the intermembrane space, building up a proton (H⁺) concentration gradient across the inner membrane.
4. Protons flow back into the matrix through ATP synthase, and this movement drives the synthesis of ATP from ADP and P_i. This coupling of the proton gradient to ATP synthesis is chemiosmosis (the chemiosmotic theory).
5. At the end of the chain, oxygen is the final (terminal) electron acceptor: it combines with the electrons and protons to form water.

$\tfrac{1}{2}O_2 + 2H^+ + 2e^- \rightarrow H_2O$

Still don't get it? · how the proton gradient makes ATP (chemiosmosis)

Think of a hydroelectric dam. A pump pushes water uphill into a high reservoir, and later the water is allowed to fall back down through a turbine, and it is the falling water that spins the turbine and generates electricity. The electricity is never made "by the pump" directly, it comes from the stored height of the water.

Now the respiration version, one step at a time:

• The reduced coenzymes bring hydrogen (as protons and electrons) to the inner membrane. The electrons are what feed the electron transfer chain.
• As electrons pass down the chain they release energy. That energy is not used to make ATP directly. Instead it is used to pump protons (H⁺) uphill, out of the matrix and into the intermembrane space. That is the "water pushed into the high reservoir".
• The protons pile up, so there is now a proton concentration gradient across the membrane. This is stored energy.
• The protons then fall back down their gradient, into the matrix, but they can only get through at one place: the enzyme ATP synthase. Flowing through it is what powers ATP synthesis. ATP synthase is the turbine.

So in the exam do not write "the electrons make ATP". The mark-scheme chain is: electrons pass down the electron transfer chain -> energy released -> protons pumped into the intermembrane space -> protons pass back through ATP synthase -> ATP made. Oxygen is only needed at the very end, as the final electron acceptor that combines with electrons and protons to form water.

The whole of respiration on one page

Per one glucose molecule, adding up all four stages:

So a single glucose yields 6 CO₂, 10 reduced NAD and 2 reduced FAD, and the reduced coenzymes then drive oxidative phosphorylation, which produces the majority of the ATP. The overall word summary is:

$C_6H_{12}O_6 + 6O_2 \rightarrow 6CO_2 + 6H_2O$

Anaerobic respiration

When there is no oxygen, the electron transfer chain stops (no final electron acceptor), so oxidative phosphorylation, the Krebs cycle and the link reaction all stop. Only glycolysis can continue, giving a net gain of just 2 ATP per glucose.

But glycolysis needs a supply of oxidised NAD to keep going, and without the electron transfer chain the reduced NAD is not being re-oxidised. Anaerobic respiration solves this by using the reduced NAD to convert pyruvate into a waste product, which regenerates the oxidised NAD:

• In animals (and bacteria): pyruvate is converted to lactate. Reduced NAD is used to reduce pyruvate, forming lactate and regenerating NAD.

  pyruvate + reduced NAD ⟶ lactate + NAD

• In plants and yeast: pyruvate is converted to ethanol and CO₂. First pyruvate loses CO₂ (decarboxylation) to form ethanal, then reduced NAD reduces ethanal to ethanol, regenerating NAD.

  pyruvate ⟶ ethanal + CO₂

  ethanal + reduced NAD ⟶ ethanol + NAD

In both pathways the point is the same: the oxidised NAD produced can be used in further glycolysis, so a small amount of ATP keeps being made without oxygen.

Still don't get it? · why anaerobic respiration bothers to make lactate or ethanol

It looks pointless: the cell takes pyruvate, a perfectly good molecule, and turns it into lactate or ethanol, which it then has to get rid of. Why?

Follow the one molecule that matters, NAD. A cell only has a limited pool of it.

• In glycolysis, NAD picks up hydrogen and becomes reduced NAD. Glycolysis cannot run without empty, oxidised NAD to load up.
• Normally the electron transfer chain unloads the reduced NAD (passing its hydrogen on towards oxygen), handing the empty NAD straight back. With no oxygen, the chain is jammed, so nothing is emptying the NAD.
• Very quickly all the NAD is full (reduced) and glycolysis grinds to a halt, so even the 2 ATP from glycolysis stops.

Making lactate or ethanol is the escape hatch. Reduced NAD dumps its hydrogen onto pyruvate (turning it into lactate, or ethanal into ethanol) purely so that the NAD is emptied and can go back to glycolysis. The lactate/ethanol is just where the spare hydrogen gets parked.

Exam wording to land on: the conversion of pyruvate to lactate/ethanol uses reduced NAD and regenerates (oxidised) NAD, which can then be used again in glycolysis so ATP production continues.

Respiratory substrates

Glucose is not the only respiratory substrate. The breakdown products of lipids and amino acids can also be respired: they enter the pathway at the Krebs cycle (lipids and amino acids are broken down into molecules that feed into the link reaction and Krebs cycle).

Lipids release more energy per gram than carbohydrates because their long hydrocarbon chains carry more hydrogen to feed oxidative phosphorylation.

Required practical 9: rate of respiration in single-celled organisms

Required practical 9 is an investigation into the effect of a named variable (for example temperature, substrate concentration or pH) on the rate of respiration of a culture of single-celled organisms such as yeast.

A redox indicator can be used to follow respiration: methylene blue (or TTC/DCPIP) is decolourised when it is reduced by hydrogen removed from the substrate during respiration, so a faster time to decolourise means a faster rate of dehydrogenase activity.

To make it a valid investigation:

• Change only the named independent variable; keep others (volume and concentration of yeast and substrate, pH, time) controlled.
• Keep temperature constant with a water bath (unless it is the variable being tested), because temperature affects the enzyme-controlled reactions of respiration.
• Include a control, for example boiled (dead) yeast, to show the result is due to living, respiring cells.
• Repeat and calculate a mean to improve reliability.

Measuring rate: the respirometer and RQ

A respirometer measures the rate of oxygen uptake by living organisms (for example germinating seeds or woodlice).

• Soda lime or potassium hydroxide in the tube absorbs the CO₂ produced, so any change in gas volume is due to oxygen uptake only. As oxygen is used, the pressure falls and the coloured manometer fluid moves towards the organisms.
• A control tube containing glass beads of the same mass (not organisms) shows that fluid movement is caused by the respiring organisms and allows for changes in room temperature/pressure.
• Rate of oxygen uptake is calculated from the distance the fluid moves in the capillary tube ($volume = \pi r^2 \times distance$) divided by time (and by mass, for a rate per gram).

The respiratory quotient (RQ) tells you which substrate is being respired:

$RQ = \frac{\text{volume of CO}_2\text{ produced}}{\text{volume of O}_2\text{ consumed}}$

An RQ of about 1.0 indicates carbohydrate is being respired; about 0.7 indicates lipid (more oxygen is needed to oxidise the long hydrocarbon chains, so relatively less CO₂ is released per O₂ used). An RQ greater than 1 shows that some anaerobic respiration is also occurring, because CO₂ is being released without a matching uptake of oxygen.

Worked examples

Model 6-mark answer: "Describe how ATP is synthesised by oxidative phosphorylation."

Each numbered point is one distinct, mark-worthy idea, in a causal order:

1. Reduced NAD (and reduced FAD) are oxidised, releasing hydrogen as protons and electrons.
2. The electrons pass along the electron transfer chain in a series of redox reactions.
3. This releases energy as the electrons move down the chain.
4. The energy is used to pump protons (H⁺) from the matrix into the intermembrane space, setting up a proton gradient.
5. Protons move back into the matrix through ATP synthase, and this drives the synthesis of ATP from ADP and P_i (chemiosmosis).
6. Oxygen is the final electron acceptor, combining with electrons and protons to form water.

The lesson: a 6-mark "describe" needs six linked points in sequence. Writing "the electrons make ATP" collapses points 2 to 5 into nothing and scores only one mark.

Worked calculation: respiratory quotient

For the aerobic respiration of glucose:

$C_6H_{12}O_6 + 6O_2 \rightarrow 6CO_2 + 6H_2O$

$RQ = \frac{\text{CO}_2\text{ produced}}{\text{O}_2\text{ used}} = \frac{6}{6} = 1.0$

Now suppose a respirometer shows an organism produces 11 cm³ of CO₂ while using 16 cm³ of O₂ in the same time:

$RQ = \frac{11}{16} = 0.69 \approx 0.7$

An RQ of about 0.7 means the organism is respiring lipid as its main substrate.

Worked calculation: rate of oxygen uptake

A respirometer holds 0.50 g of germinating seeds. The manometer fluid moves 60 mm in 5 minutes along a capillary tube of radius 0.5 mm.

$\text{volume of O}_2 = \pi r^2 \times distance = \pi \times (0.5)^2 \times 60 = 47.1\ \text{mm}^3$

$\text{rate} = \frac{47.1}{0.50 \times 5} = 18.8\ \text{mm}^3\,\text{g}^{-1}\,\text{min}^{-1}$

The common lost mark here is forgetting to divide by both the mass (to get per gram) and the time.

Common exam mistakes

• Writing that respiration "produces" or "makes" energy. Energy is released (from respiratory substrates); it is never created. This is the single most common error examiners report.
• Writing NADP instead of NAD. NADP belongs to photosynthesis; respiration uses NAD (and FAD). Using NADP is penalised.
• Saying glycolysis requires oxygen. Glycolysis is anaerobic and occurs in the cytoplasm; oxygen is only needed later, at the electron transfer chain.
• Confusing net and gross ATP in glycolysis: 4 ATP are made but 2 are used, so the net gain is 2.
• Forgetting that the link reaction and Krebs cycle each occur twice per glucose (because glycolysis makes two pyruvate), so halving the totals.
• In the link reaction, saying reduced NAD is used rather than formed, or that ATP is used or that pyruvate is reduced. Pyruvate is oxidised, forming reduced NAD.
• In anaerobic respiration, saying that converting pyruvate to lactate forms reduced NAD. It is the opposite: reduced NAD is used and oxidised NAD is regenerated so glycolysis can continue. You must state that the regenerated NAD is reused in glycolysis, not just that "glycolysis continues".
• Saying oxygen is used directly in the Krebs cycle. Oxygen acts only as the final electron acceptor at the end of the electron transfer chain.
• Explaining oxidative phosphorylation as "electrons make ATP" without the proton gradient. The marks are for: electrons down the chain, energy released, protons pumped into the intermembrane space, protons back through ATP synthase.
• Writing that NAD accepts only protons. NAD accepts hydrogen (protons and electrons).
• With a respirometer, forgetting to state that KOH or soda lime absorbs CO₂ so the volume change is due to O₂ uptake only, and forgetting the control tube of glass beads.
• Not knowing that RQ for aerobic respiration of glucose is 1, or that an RQ above 1 indicates anaerobic respiration is also happening.

Key definitions

• Glycolysis - the first stage of respiration, occurring in the cytoplasm, in which glucose is split into two molecules of pyruvate, producing a net gain of ATP and reduced NAD; it is anaerobic.
• Decarboxylation - the removal of a carbon atom from a molecule as carbon dioxide.
• Substrate-level phosphorylation - the formation of ATP by the direct transfer of a phosphate group to ADP from a substrate molecule (in glycolysis and the Krebs cycle).
• Oxidative phosphorylation - the synthesis of ATP using energy released as electrons are transferred down the electron transfer chain from reduced coenzymes.
• Chemiosmosis - the movement of protons down their concentration gradient across the inner mitochondrial membrane, through ATP synthase, providing the energy to synthesise ATP.
• Coenzyme - a molecule that transfers a chemical group from one reaction to another; NAD and FAD are coenzymes that carry hydrogen (protons and electrons).
• Respiratory substrate - an organic molecule that is oxidised in respiration to release energy for the synthesis of ATP.
• Respiratory quotient (RQ) - the volume of carbon dioxide produced divided by the volume of oxygen consumed in the same time during respiration.

Specification

• I can state that respiration produces ATP.
• I can describe glycolysis as the anaerobic first stage in the cytoplasm: phosphorylation of glucose to glucose phosphate using ATP, production of triose phosphate, and oxidation of triose phosphate to pyruvate with a net gain of ATP and reduced NAD.
• I can explain that in anaerobic respiration pyruvate is converted to ethanol or lactate using reduced NAD, and that the oxidised NAD produced is reused in glycolysis.
• I can state that in aerobic respiration pyruvate enters the mitochondrial matrix by active transport.
• I can describe the link reaction: pyruvate is oxidised to acetate (producing reduced NAD) and acetate combines with coenzyme A to form acetylcoenzyme A.
• I can describe how acetylcoenzyme A reacts with a 4-carbon molecule to form a 6-carbon molecule that enters the Krebs cycle, releasing coenzyme A.
• I can explain that the Krebs cycle generates reduced coenzymes and ATP by substrate-level phosphorylation through a series of oxidation-reduction reactions, and that carbon dioxide is lost.
• I can explain that ATP synthesis by oxidative phosphorylation is associated with electron transfer down the electron transfer chain and the passage of protons across inner mitochondrial membranes, catalysed by ATP synthase (chemiosmotic theory).
• I can state that other respiratory substrates include the breakdown products of lipids and amino acids, which enter the Krebs cycle.
• I can carry out required practical 9: investigating the effect of a named variable on the rate of respiration of cultures of single-celled organisms.

Related notes

• ATP
• Cell structure (the mitochondrion)
• Photosynthesis
• Energy and ecosystems