Welcome to Respiration and Respiratory Quotient!
Welcome to one of the most fundamental chapters in your IA2 Biology course! Everything your body does—from contracting a muscle to sending a nerve impulse—requires energy. That energy is made available through cellular respiration. Don't worry if biochemical pathways seem daunting at first; we will break down each stage into simple, bite-sized steps so you can master both the theory and the required calculations with confidence.
Note on syllabus boundaries: In this chapter, we focus specifically on aerobic respiration, lactate metabolism, the Respiratory Quotient (RQ), and our core practicals on respirometry and redox indicators. Related topics such as muscle contraction, ventilation control, and kidney function are explored in companion chapters within Unit 5.
---1. Overview of Aerobic Respiration
Aerobic respiration is not a single rapid combustion reaction. Instead, it is a stepped process controlled by intracellular enzymes. Breaking the oxidation of glucose down into many small, enzyme-catalysed steps allows cells to control energy release and efficiently trap it by synthesising adenosine triphosphate (ATP), rather than releasing all energy dangerously at once as heat.
Aerobic respiration proceeds in four linked stages:
1. Glycolysis (occurs in the cytoplasm)
2. The Link Reaction (occurs in the mitochondrial matrix)
3. The Krebs Cycle (occurs in the mitochondrial matrix)
4. Oxidative Phosphorylation (occurs at the inner mitochondrial membrane)
Key Takeaway: Glycolysis takes place in the cell cytoplasm and does not require oxygen. All subsequent stages take place inside the mitochondria and depend directly or indirectly on the presence of oxygen.
---2. Stage 1: Glycolysis (In the Cytoplasm)
Glycolysis literally means "sugar splitting". It is the only stage of cellular respiration that takes place outside the mitochondria, occurring directly in the fluid cytoplasm of the cell.
Step-by-Step Pathway of Glycolysis
1. Hexose Phosphorylation: The starting stable 6-carbon sugar (hexose, such as glucose) is phosphorylated by adding phosphate groups from ATP. This activates the sugar and makes it more chemically reactive.
2. Lysis: The phosphorylated hexose is split into two 3-carbon intermediate compounds.
3. Oxidation and Formation of Reduced Coenzyme: Hydrogen atoms are removed from these 3-carbon compounds and transferred to the coenzyme NAD, converting it into reduced NAD.
4. Substrate-Level Phosphorylation: Phosphate groups are transferred directly from intermediate compounds to ADP, generating ATP without an electron transport chain. This direct transfer is termed substrate-level phosphorylation.
5. End Product: The final 3-carbon product formed is pyruvate.
Net Yield per Glucose Molecule in Glycolysis:
• \(2\) molecules of pyruvate
• \(2\) molecules of ATP (net gain: \(2\) invested during phosphorylation, \(4\) produced via substrate-level phosphorylation)
• \(2\) molecules of reduced NAD
Did you know? Because glycolysis requires no oxygen and no membrane-bound organelles, it is considered one of the most primitive metabolic pathways on Earth, shared by virtually all living organisms!
Key Takeaway: Glycolysis phosphorylates hexose, yields a net gain of \(2\text{ ATP}\) by substrate-level phosphorylation, reduces NAD, and produces \(2\) molecules of pyruvate in the cytoplasm.
---3. Anaerobic Conditions: The Fate of Pyruvate and Lactate
What happens when oxygen is unavailable or in short supply, such as during intense muscular exertion? (For muscle contraction mechanics, see the Muscles and Movement chapter).
Formation of Lactate
Without oxygen to act as the terminal electron acceptor in mitochondria, the electron transport chain stops running, and reduced NAD cannot deposit its hydrogens there. Soon, all intracellular NAD would become fully reduced, bringing glycolysis to a halt and stopping all ATP production.
To keep glycolysis running and producing its small net yield of \(2\text{ ATP}\):
• Pyruvate accepts hydrogen atoms directly from reduced NAD.
• Pyruvate is reduced to lactate.
• This oxidises reduced NAD back to NAD, freeing it up to return to glycolysis and accept more hydrogens.
The Fate of Lactate
Lactate cannot accumulate indefinitely because it causes tissue acidosis and fatigue. Its fate involves:
• Lactate diffuses out of active cells into the blood and is transported to the liver.
• In the liver, when oxygen becomes available again, lactate can be converted back into pyruvate.
• This pyruvate can then enter the aerobic link reaction and Krebs cycle, or be converted into glucose/glycogen for storage.
Key Takeaway: Lactate production regenerates oxidised NAD so glycolysis can continue producing ATP anaerobically. Lactate is later converted back to pyruvate in the liver when oxygen is restored.
---4. Moving into the Mitochondria: Link Reaction and Krebs Cycle
Under aerobic conditions, pyruvate produced in the cytoplasm is actively transported across the mitochondrial membranes into the mitochondrial matrix.
The Link Reaction
The link reaction connects glycolysis to the Krebs cycle:
• Decarboxylation: A carbon atom is removed from 3-carbon pyruvate and released as a molecule of carbon dioxide (\(\text{CO}_2\)).
• Dehydrogenation (Oxidation): Hydrogen is removed and accepted by NAD to form reduced NAD.
• The resulting 2-carbon acetyl group binds with coenzyme A to enter the Krebs cycle as acetyl coenzyme A.
• Per glucose molecule (\(2\) pyruvates), the link reaction produces: \(2\text{ CO}_2\), \(2\) reduced NAD, and \(2\) acetyl groups.
The Krebs Cycle
The Krebs cycle is a cyclic series of oxidation-reduction reactions occurring within the mitochondrial matrix:
• The 2-carbon acetyl fragment combines with a 4-carbon compound to form a 6-carbon compound.
• Through successive steps, the compound undergoes two separate decarboxylations, releasing \(2\) molecules of \(\text{CO}_2\).
• It undergoes oxidation steps transferring hydrogens to yield reduced NAD and reduced FAD.
• A molecule of ATP is produced directly via substrate-level phosphorylation.
• The starting 4-carbon compound is regenerated to begin the cycle again.
Summary Yield of Krebs Cycle per Glucose Molecule (2 turns):
• \(4\text{ CO}_2\)
• \(6\) reduced NAD
• \(2\) reduced FAD
• \(2\text{ ATP}\) (by substrate-level phosphorylation)
Key Takeaway: The link reaction and Krebs cycle occur entirely in the mitochondrial matrix. They release carbon as \(\text{CO}_2\) via decarboxylation and harvest high-energy hydrogen atoms via reduced NAD and reduced FAD.
---5. Stage 4: Oxidative Phosphorylation and Chemiosmosis
This final stage delivers the greatest payoff of ATP. It takes place at the inner mitochondrial membrane (cristae).
The Electron Transport Chain (ETC)
1. Donating Electrons: Reduced NAD and reduced FAD pass their hydrogen atoms to the electron transport chain embedded in the inner mitochondrial membrane. The hydrogens split into protons (\(\text{H}^+\)) and high-energy electrons (\(e^-\)). NAD and FAD are regenerated and return to the matrix.
2. Electron Flow: Electrons travel along a sequence of electron carriers in redox reactions, releasing energy as they transfer from higher to lower energy levels.
3. Proton Pumping: The energy released by these electrons is used to actively pump protons (\(\text{H}^+\)) from the mitochondrial matrix across the inner membrane into the intermembrane space.
4. Electrochemical Gradient: This creates a high concentration of protons in the intermembrane space compared to the matrix, setting up a steep proton (electrochemical) gradient.
Chemiosmosis and ATP Synthase
• The inner membrane is impermeable to protons, so they can only diffuse back into the matrix through a specialised channel enzyme: ATP synthase.
• The movement of protons down their electrochemical gradient through ATP synthase provides the energy to drive the phosphorylation of ADP and inorganic phosphate into ATP. This mechanism is called chemiosmosis.
Oxygen as the Final Electron Acceptor
At the end of the electron transport chain, electrons, protons from the matrix, and molecular oxygen combine:
\(4\text{H}^+ + 4e^- + \text{O}_2 \longrightarrow 2\text{H}_2\text{O}\)
Without oxygen to remove electrons from the final carrier, electron transport stops, the proton gradient dissipates, and oxidative phosphorylation ceases.
Key Takeaway: Oxidative phosphorylation uses electrons from reduced NAD/FAD along the ETC to pump protons. The return of protons through ATP synthase synthesises ATP by chemiosmosis, with oxygen acting as the final electron acceptor to form water.
---6. The Respiratory Quotient (RQ)
The Respiratory Quotient (RQ) is a dimensionless ratio that gives insight into the respiratory substrate being metabolised by an organism, as well as whether respiration is aerobic or anaerobic.
Formula
The syllabus defines the Respiratory Quotient as:
\(\text{RQ} = \frac{\text{carbon dioxide produced}}{\text{oxygen consumed}}\)
Both quantities must be measured over the same time period and in identical units of volume or moles.
Typical RQ Values for Different Substrates
• Carbohydrates (e.g. Glucose): \(\text{RQ} = 1.0\)
\(\text{C}_6\text{H}_{12}\text{O}_6 + 6\text{O}_2 \longrightarrow 6\text{CO}_2 + 6\text{H}_2\text{O}\)
\(\text{RQ} = \frac{6\text{ CO}_2}{6\text{ O}_2} = 1.0\)
• Lipids (Fatty acids): \(\text{RQ} \approx 0.7\)
Lipids contain a much higher proportion of hydrogen-to-carbon and very little oxygen within their molecular structure. Consequently, they require substantially more oxygen molecules to oxidise each carbon atom to \(\text{CO}_2\), resulting in a lower RQ value.
• Proteins / Amino acids: \(\text{RQ} \approx 0.8 - 0.9\)
Interpreting Anomalous RQ Values
• RQ significantly greater than \(1.0\): Indicates that some anaerobic respiration is taking place (since carbon dioxide is still being released during decarboxylation without corresponding oxygen consumption), or that carbohydrates are being actively converted into fat storage.
• Common Exam Mistake: Forgetting that RQ has no units! Always present RQ as a single decimal number (e.g., \(0.72\)), not as a fraction or with units.
Key Takeaway: \(\text{RQ} = \frac{\text{volume of }\text{CO}_2\text{ produced}}{\text{volume of }\text{O}_2\text{ consumed}}\). Carbohydrate oxidation yields an RQ of \(1.0\), lipids around \(0.7\), and proteins around \(0.8\text{–}0.9\). Values above \(1.0\) indicate anaerobic respiration.
---7. Core Practical Skills in Respiration
Core Practical 15: Investigating Respiration in Yeast with an Artificial Hydrogen Carrier
• Principle: An artificial redox indicator / hydrogen carrier (such as methylene blue or DCPIP) acts as a substitute electron/hydrogen acceptor. When reduced by hydrogens released during yeast dehydrogenase activity, the indicator undergoes a distinct colour change (e.g. blue to colourless).
• Dependent Variable: Rate of respiration, calculated as \(\text{rate} = \frac{1}{\text{time taken for colour change (s)}}\).
• Key Controls: Temperature (using a thermostatically controlled water bath), yeast suspension concentration and volume, substrate concentration (e.g. glucose), and pH (using buffer solutions).
Core Practical 16: Using a Simple Respirometer
A simple respirometer measures gas exchange in living respiring material, such as germinating seeds or small invertebrates (treated with appropriate ethical care).
Apparatus and Method:
1. Measuring Oxygen Consumption:
• The organism is placed in a sealed chamber connected to a calibrated capillary tube containing a droplet of coloured liquid.
• A chemical absorbent (such as potassium hydroxide or soda lime) is placed in the tube to absorb all \(\text{CO}_2\) produced.
• As the organism respires, it consumes \(\text{O}_2\). The \(\text{CO}_2\) produced is absorbed by the chemical, reducing the gas volume and pressure inside the chamber.
• The coloured liquid droplet moves toward the organism. The distance moved allows calculation of oxygen volume consumed: \(\text{Volume} = \pi r^2 d\) (where \(r\) is capillary bore radius, \(d\) is distance moved).
2. Determining Respiratory Quotient (RQ):
• The experiment is repeated under identical conditions without the \(\text{CO}_2\) absorbent.
• The movement of the droplet now represents the difference between \(\text{O}_2\) consumed and \(\text{CO}_2\) produced.
• If \(\text{RQ} = 1.0\), \(\text{O}_2\) consumed equals \(\text{CO}_2\) produced, so the droplet does not move.
• If \(\text{RQ} < 1.0\) (e.g. lipid), more \(\text{O}_2\) is absorbed than \(\text{CO}_2\) released; volume decreases, and the droplet moves toward the chamber.
• By comparing the net volume change without absorbent to the oxygen consumption measured with absorbent, the volume of \(\text{CO}_2\) released is determined: \(\text{CO}_2\text{ produced} = \text{volume change without absorbent} + \text{oxygen consumed}\).
Key Controlled Variables in Respirometry:
• Temperature: Changes in ambient temperature cause air inside the sealed tube to expand or contract according to gas laws. Respirometers must be immersed in a water bath to maintain constant temperature.
• Atmospheric Pressure: A control tube without living organisms (containing equal volume of inert glass beads) is often run concurrently as a thermobarometer to correct for changes in atmospheric pressure.
Key Takeaway: In respirometry, soda lime absorbs \(\text{CO}_2\) to allow measurement of \(\text{O}_2\) uptake alone. Repeating without soda lime allows \(\text{CO}_2\) output and RQ to be determined.
---Quick Summary & Checkpoint
• Respiration is stepped: Controlled by intracellular enzymes to release energy progressively.
• Glycolysis: In cytoplasm; phosphorylates hexose, yields net \(2\text{ ATP}\) (substrate-level phosphorylation), \(2\) reduced NAD, and \(2\) pyruvates.
• Anaerobic conditions: Pyruvate reduces to lactate, regenerating NAD so glycolysis continues.
• Link & Krebs: In mitochondrial matrix; decarboxylation releases \(\text{CO}_2\), dehydrogenation reduces NAD and FAD.
• Oxidative phosphorylation: In inner mitochondrial membrane; electron transport chain pumps \(\text{H}^+\), chemiosmosis through ATP synthase generates ATP, and \(\text{O}_2\) acts as the final electron acceptor.
• RQ calculation: \(\text{RQ} = \frac{\text{carbon dioxide produced}}{\text{oxygen consumed}}\). (\(1.0\) for carbohydrates, \(0.7\) for lipids, \(>1.0\) for anaerobic respiration).