Respiration (In Depth)

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Cellular respiration is the controlled release of energy from organic molecules ( glucose) to produce ATP. It is an exergonic, enzyme-catalysed process that occurs in every living cell.

The overall equation for aerobic respiration of glucose:

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

$\Delta G = -2870\ \mathrm{kJ\ mol^{-1}}$

However, not all of this energy is captured as ATP. Approximately 40% is captured (theoretical maximum approximately 38 ATP; actual yield approximately 30—32 ATP). The remainder is released as heat, which maintains body temperature in endotherms.

Total per glucose molecule: approximately 30—32 ATP (varies between cell types and organisms).

Glycolysis (“sugar splitting”) is the first stage of both aerobic and anaerobic respiration. It occurs in the cytoplasm and does not require oxygen. One molecule of glucose ($\mathrm{C_6H_{12}O_6}$6-carbon) is converted into two molecules of pyruvate ($\mathrm{CH_3COCOO^-}$3-carbon).

Phase 1: Energy Investment (uses 2 ATP)

Step 1: Phosphorylation of glucose. Glucose is phosphorylated by hexokinase (or glucokinase in the liver), using ATP:

$\mathrm{Glucose + ATP \to glucose\text{-}6\text{-}phosphate + ADP}$

This traps glucose inside the cell (phosphorylated glucose cannot cross the cell membrane) and makes it more reactive.

Step 2: Isomerisation. Glucose-6-phosphate is converted to fructose-6-phosphate by phosphoglucose isomerase. This rearranges the molecule to enable the formation of two 3-carbon compounds later.

Step 3: Second phosphorylation. Fructose-6-phosphate is phosphorylated by phosphofructokinase (PFK), using a second ATP:

$\mathrm{Fructose\text{-}6\text{-}phosphate + ATP \to fructose\text{-}1,6\text{-}bisphosphate + ADP}$

PFK is a key regulatory enzyme: it is allosterically inhibited by ATP and citrate (signalling high energy status) and activated by AMP and fructose-2,6-bisphosphate (signalling low energy status).

Step 4: Cleavage. Fructose-1,6-bisphosphate is cleaved by aldolase into two 3-carbon molecules: glyceraldehyde-3-phosphate (G3P, also called triose phosphate, TP) and dihydroxyacetone phosphate (DHAP).

Step 5: Isomerisation. DHAP is converted to G3P by triose phosphate isomerase. From this point, both molecules follow the same pathway. All subsequent steps occur twice per glucose molecule.

Phase 2: Energy Payoff (produces 4 ATP + 2 NADH)

Step 6: Oxidation and phosphorylation. Each G3P is oxidised by triose phosphate dehydrogenase, which transfers two electrons and a proton to $\mathrm{NAD^+}$Forming NADH. A phosphate group is added from inorganic phosphate ($P_i$), producing 1,3-bisphosphoglycerate:

$\mathrm{G3P + NAD^+ + P_i \to 1,3\text{-}BPG + NADH + H^+}$

Step 7: Substrate-level phosphorylation (first). Phosphoglycerate kinase transfers a phosphate group from 1,3-BPG to ADP, producing ATP and 3-phosphoglycerate:

$\mathrm{1,3\text{-}BPG + ADP \to 3\text{-}phosphoglycerate + ATP}$

This occurs twice per glucose, producing 2 ATP (recovering the 2 ATP invested in steps 1 and 3).

Step 8: Rearrangement. 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase.

Step 9: Dehydration. 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP) by enolase, releasing one molecule of $\mathrm{H_2O}$ per molecule (2 per glucose):

$\mathrm{2\text{-}phosphoglycerate \to PEP + H_2O}$

Step 10: Substrate-level phosphorylation (second). Pyruvate kinase transfers the phosphate from PEP to ADP, producing ATP and pyruvate:

$\mathrm{PEP + ADP \to pyruvate + ATP}$

This occurs twice per glucose, producing 2 additional ATP.

$\Delta G^\circ_{\text{glycolysis}} = -63\ \mathrm{kJ\ mol^{-1}}$

The reaction is exergonic overall but contains both endergonic and exergonic steps. The energy-investment steps (steps 1 and 3) are endergonic and are coupled to the exergonic hydrolysis of ATP. The energy-payoff steps (7 and 10) are exergonic and generate ATP by substrate-level phosphorylation.

The link reaction (also called the pyruvate dehydrogenase reaction) converts pyruvate from glycolysis into acetyl coenzyme A (acetyl CoA), which enters the Krebs cycle. It occurs in the mitochondrial matrix and requires oxygen indirectly (the Krebs cycle and oxidative phosphorylation that follow require oxygen to regenerate $\mathrm{NAD^+}$ and FAD).

The reaction occurs twice per glucose molecule (one per pyruvate):

$\mathrm{Pyruvate + CoA + NAD^+ \to acetyl\ CoA + CO_2 + NADH + H^+}$

Key points:

• Pyruvate (3-carbon) loses one carbon as $\mathrm{CO_2}$ (decarboxylation).
• The remaining 2-carbon fragment (acetyl group) is transferred to coenzyme A, forming acetyl CoA.
• $\mathrm{NAD^+}$ is reduced to $\mathrm{NADH}$.
• The reaction is catalysed by the pyruvate dehydrogenase complex, a large multi-enzyme complex.

The Krebs cycle (also called the citric acid cycle or tricarboxylic acid cycle, TCA cycle) is a series of enzyme-catalysed reactions in the mitochondrial matrix. It completes the oxidation of acetyl CoA, releasing $\mathrm{CO_2}$ and transferring high-energy electrons to $\mathrm{NAD^+}$FAD, and ATP.

The cycle turns twice per glucose molecule (one turn per acetyl CoA).

Step 1: Formation of citrate. Acetyl CoA (2-carbon) combines with oxaloacetate (4-carbon) to form citrate (6-carbon). CoA is released and recycled. Catalysed by citrate synthase.

$\mathrm{Acetyl\ CoA + oxaloacetate + H_2O \to citrate + CoA + H^+}$

Step 2: Isomerisation to isocitrate. Citrate is converted to isocitrate by aconitase. This rearrangement makes the molecule more reactive for the next step.

Step 3: First oxidative decarboxylation. Isocitrate is oxidised and decarboxylated by isocitrate dehydrogenase, producing $\alpha$-ketoglutarate (5-carbon), $\mathrm{CO_2}$And NADH. This is a key regulatory step.

$\mathrm{Isocitrate + NAD^+ \to \alpha\text{-}ketoglutarate + CO_2 + NADH + H^+}$

Step 4: Second oxidative decarboxylation. $\alpha$-Ketoglutarate is oxidised and decarboxylated by the $\alpha$-ketoglutarate dehydrogenase complex, producing succinyl CoA (4-carbon), $\mathrm{CO_2}$And NADH.

$\mathrm{\alpha\text{-}Ketoglutarate + CoA + NAD^+ \to succinyl\ CoA + CO_2 + NADH + H^+}$

Step 5: Substrate-level phosphorylation. Succinyl CoA is converted to succinate by succinyl thiokinase (succinyl CoA synthetase). The energy released by cleaving the thioester bond in succinyl CoA is used to phosphorylate GDP to GTP, which is equivalent to ATP:

$\mathrm{Succinyl\ CoA + GDP + P_i \to succinate + GTP + CoA + H^+}$

Step 6: Oxidation of succinate. Succinate is oxidised to fumarate by succinate dehydrogenase (an integral membrane protein embedded in the inner mitochondrial membrane, directly feeding electrons to the electron transport chain via FAD). FAD is reduced to $\mathrm{FADH_2}$.

$\mathrm{Succinate + FAD \to fumarate + FADH_2}$

Step 7: Hydration. Fumarate is hydrated to malate by fumarase, adding $\mathrm{H_2O}$:

$\mathrm{Fumarate + H_2O \to malate}$

Step 8: Oxidation to oxaloacetate. Malate is oxidised to oxaloacetate by malate dehydrogenase, reducing $\mathrm{NAD^+}$ to NADH. This regenerates oxaloacetate, which can accept another acetyl CoA, continuing the cycle.

$\mathrm{Malate + NAD^+ \to oxaloacetate + NADH + H^+}$

• NADH: carries electrons to the electron transport chain. Each NADH ultimately generates approximately 2.5 ATP.
• $\mathrm{FADH_2}$: carries electrons to the electron transport chain (enters at a later point). Each $\mathrm{FADH_2}$ generates approximately 1.5 ATP.
• $\mathrm{CO_2}$: released as a waste product; excreted via the lungs.
• GTP/ATP: used directly by the cell.

Oxidative phosphorylation is the final stage of aerobic respiration. It occurs on the inner mitochondrial membrane (cristae) and requires oxygen as the final electron acceptor. It produces the vast majority of ATP from the oxidation of glucose.

The electron transport chain is a series of membrane-bound protein complexes and mobile electron carriers embedded in the inner mitochondrial membrane. Electrons from NADH and $\mathrm{FADH_2}$ pass through the chain in a series of redox reactions, releasing energy that is used to pump protons across the inner membrane.

Components of the ETC (in order):

1. Complex I (NADH dehydrogenase): accepts electrons from NADH and passes them to ubiquinone (coenzyme Q, Q). Pumps $4\ \mathrm{H^+}$ from the matrix to the intermembrane space.

2. Complex II (succinate dehydrogenase): accepts electrons from $\mathrm{FADH_2}$ (produced in the Krebs cycle) and passes them to ubiquinone. Does not pump protons.

3. Ubiquinone (coenzyme Q): a lipid-soluble mobile electron carrier that shuttles electrons from Complexes I and II to Complex III.

4. Complex III (cytochrome $bc_1$): receives electrons from ubiquinone and passes them to cytochrome c. Pumps $4\ \mathrm{H^+}$.

5. Cytochrome c: a small peripheral membrane protein that shuttles electrons from Complex III to Complex IV.

6. Complex IV (cytochrome c oxidase): receives electrons from cytochrome c and transfers them to molecular oxygen ($\mathrm{O_2}$), the final electron acceptor. Oxygen combines with electrons and protons to form water:

$\mathrm{O_2 + 4e^- + 4H^+ \to 2H_2O}$

Complex IV pumps $2\ \mathrm{H^+}$.

The chemiosmotic theory explains how the energy from electron transport is used to synthesise ATP.

1. As electrons pass through Complexes I, III, and IV, protons ($\mathrm{H^+}$) are actively pumped from the mitochondrial matrix to the intermembrane space.
2. This creates a proton gradient across the inner membrane: high $\mathrm{H^+}$ concentration in the intermembrane space, low in the matrix. This is both a chemical gradient (difference in $\mathrm{H^+}$ concentration) and an electrical gradient (difference in charge).
3. The combined electrochemical gradient is called the proton motive force (PMF):

$\mathrm{PMF} = \Delta\Psi - \frac{2.303RT}{F}\Delta\mathrm{pH}$

Where $\Delta\Psi$ is the membrane potential (approximately $150$—$180\ \mathrm{mV}$) and $\Delta\mathrm{pH}$ is the pH difference across the membrane (approximately 0.5—1.0 unit).

4. Protons can only return to the matrix through ATP synthase (Complex V), a transmembrane protein complex that acts as a molecular motor.
5. As protons flow through ATP synthase, the flow drives the rotation of a rotor, which induces conformational changes in the catalytic domains that synthesise ATP from ADP and $P_i$:

$\mathrm{ADP + P_i \to ATP}$

Proton pumping summary:

Approximately 3—4 protons must flow through ATP synthase to synthesise one ATP molecule (the exact number depends on the organism and conditions; a commonly cited value is $4\ \mathrm{H^+}$ per ATP: 3 for ATP synthesis and 1 for phosphate transport).

Note: the actual yield in most cells is approximately 30—32 ATP per glucose molecule, lower than the theoretical maximum due to proton leakage across the inner membrane, the cost of transporting pyruvate and ADP into the mitochondria, and other inefficiencies.

Oxygen is the final electron acceptor in the electron transport chain. Without oxygen:

• Electrons cannot pass through Complex IV (they back up through the chain).
• Proton pumping stops, the proton gradient dissipates, and ATP synthase stops.
• NADH and $\mathrm{FADH_2}$ cannot be reoxidised to $\mathrm{NAD^+}$ and FAD.
• Without $\mathrm{NAD^+}$The Krebs cycle and glycolysis (specifically step 6) stop.

This is why cells must switch to anaerobic respiration in the absence of oxygen.

In the absence of oxygen, pyruvate from glycolysis cannot enter the mitochondria for the link reaction and Krebs cycle. Instead, pyruvate is converted to lactate (lactic acid) by the enzyme lactate dehydrogenase:

$\mathrm{Pyruvate + NADH + H^+ \to lactate + NAD^+}$

The critical purpose of this reaction is to regenerate $\mathrm{NAD^+}$, allowing glycolysis to continue. Without $\mathrm{NAD^+}$Glycolysis would stop at step 6, and the cell would have no ATP production.

ATP yield from anaerobic respiration: 2 ATP per glucose (only from glycolysis; the link reaction, Krebs cycle, and oxidative phosphorylation do not occur).

Yeast and some plant cells carry out alcoholic fermentation:

$\mathrm{Pyruvate \xrightarrow{\text{decarboxylase}} ethanal + CO_2}$

$\mathrm{Ethanal + NADH + H^+ \xrightarrow{\text{alcohol dehydrogenase}} ethanol + NAD^+}$

Products: ethanol and $\mathrm{CO_2}$. ATP yield: 2 ATP per glucose.

This reaction is exploited in brewing (ethanol production) and bread-making ($\mathrm{CO_2}$ production causes dough to rise).

Different respiratory substrates release different amounts of energy per unit mass:

Lipids contain approximately 2.5 times more energy per gram than carbohydrates because they have a higher proportion of $\mathrm{C{-}H}$ bonds (which release more energy when oxidised than \mathrm{C{-}O bonds). This is why lipids are preferred for long-term energy storage.

The respiratory quotient (RQ) is the ratio of $\mathrm{CO_2}$ produced to $\mathrm{O_2}$ consumed:

$RQ = \frac{\text{Volume of }\mathrm{CO_2}\text{ produced}}{\text{Volume of }\mathrm{O_2}\text{ consumed}}$

For different substrates:

Interpreting RQ values:

• $RQ \approx 1.0$: the organism is primarily respiring carbohydrates.
• $RQ \approx 0.7$: the organism is primarily respiring lipids.
• $RQ > 1.0$: the organism is respiring anaerobically (producing $\mathrm{CO_2}$ without consuming $\mathrm{O_2}$) or performing short bursts of intense exercise (where anaerobic respiration supplements aerobic respiration).
• $RQ \approx 0.8$: the organism is using a mixture of substrates.

A respirometer experiment shows that a germinating seedling consumes $24.0\ \mathrm{cm^3}$ of $\mathrm{O_2}$ and produces $17.0\ \mathrm{cm^3}$ of $\mathrm{CO_2}$ in 30 minutes.

$RQ = \frac{17.0}{24.0} = 0.71$

An RQ of 0.71 indicates that the seedling is primarily respiring lipids (using energy stores in the seed).

As temperature increases from $0\ ^\circ\mathrm{C}$ to the optimum (approximately $35$—$40\ ^\circ\mathrm{C}$), the rate of respiration increases due to increased kinetic energy and more frequent enzyme-substrate collisions. Above the optimum, enzymes denature and the rate decreases sharply. This follows the same pattern as any enzyme-catalysed reaction.

Increasing glucose concentration increases the respiration rate up to a point. At very high concentrations, the rate plateaus as the enzymes involved become saturated (limited by enzyme concentration, not substrate).

Aerobic respiration rate increases with $\mathrm{O_2}$ concentration until the rate plateaus. Below a critical $\mathrm{O_2}$ concentration, aerobic respiration cannot meet energy demands, and anaerobic pathways are activated.

High $\mathrm{CO_2}$ concentrations can inhibit respiration (competitive inhibition of enzymes such as RuBisCO in photosynthesis; in respiration, high $\mathrm{CO_2}$ can reduce enzyme activity in some tissues).

A respirometer measures the rate of respiration by measuring either:

• The consumption of $\mathrm{O_2}$ (volume of $\mathrm{O_2}$ absorbed per unit time).
• The production of $\mathrm{CO_2}$ (volume of $\mathrm{CO_2}$ released per unit time).

A simple respirometer consists of:

• A sealed chamber containing the respiring organism (e.g., germinating seeds or small invertebrates).
• A tube connecting the chamber to a manometer (U-tube containing coloured liquid) or a syringe.
• A chemical to absorb $\mathrm{CO_2}$ (e.g., soda lime or potassium hydroxide solution).

Procedure:

1. The organism is placed in the chamber with soda lime to absorb $\mathrm{CO_2}$.
2. As the organism respires, it consumes $\mathrm{O_2}$ and produces $\mathrm{CO_2}$.
3. The $\mathrm{CO_2}$ is absorbed by the soda lime, creating a pressure drop in the chamber (gas volume decreases because $\mathrm{O_2}$ is consumed but $\mathrm{CO_2}$ is removed).
4. The pressure drop causes the coloured liquid in the manometer to move towards the chamber.
5. The distance moved by the liquid is proportional to the volume of $\mathrm{O_2}$ consumed.

Worked Example. In a respirometer experiment, the coloured liquid in the manometer moves $28\ \mathrm{mm}$ in 10 minutes. The manometer tube has an internal diameter of $1.0\ \mathrm{mm}$.

Cross-sectional area of tube $= \pi r^2 = \pi \times (0.5)^2 = 0.785\ \mathrm{mm^2}$.

Volume of $\mathrm{O_2}$ consumed $= 0.785 \times 28 = 22.0\ \mathrm{mm^3} = 0.022\ \mathrm{cm^3}$.

Rate of $\mathrm{O_2}$ consumption $= \frac{0.022}{10} = 0.0022\ \mathrm{cm^3\ min^{-1}}$.

• Control: a respirometer with dead organisms (boiled seeds) to account for any non-respiratory changes in gas volume (e.g., temperature fluctuations).
• Temperature control: the respirometer must be placed in a water bath at constant temperature to prevent thermal expansion or contraction of gases.
• Volume correction: the readings must be corrected for the fact that the organism produces $\mathrm{CO_2}$ that the soda lime absorbs. Without soda lime, the net gas volume change would be smaller (because $\mathrm{CO_2}$ production partially offsets $\mathrm{O_2}$ consumption).

The overall free energy change for aerobic respiration of glucose is approximately $-2870\ \mathrm{kJ\ mol^{-1}}$. This energy is released in stages, with each stage releasing a specific amount:

The energy released at each stage is not directly captured as ATP. The efficiency of energy capture varies:

• Substrate-level phosphorylation captures approximately 40% of the energy released in glycolysis and the Krebs cycle as ATP.
• Oxidative phosphorylation captures approximately 40% of the energy released by electron transport as ATP.
• The remaining 60% at each stage is lost as heat, maintaining body temperature.

Overall efficiency of aerobic respiration:

$\text{Efficiency} = \frac{\text{Energy captured as ATP}}{\text{Total energy released}} = \frac{30 \times 30.5}{2870} = \frac{915}{2870} = 31.9\%$

This means approximately 68% of the energy in glucose is lost as heat. While this seems inefficient, it is sufficient for the metabolic demands of most organisms because glucose is continuously available.

Respiration demonstrates thermodynamic coupling: an exergonic reaction (the oxidation of substrates) is coupled to an endergonic reaction (the phosphorylation of ADP to ATP). The coupling agent is the proton gradient: the energy released by electron transport is temporarily stored as an electrochemical gradient, which then drives ATP synthesis.

This coupling is not 100% efficient because:

• Some protons leak back across the inner membrane without passing through ATP synthase (proton leak).
• The proton gradient is also used for other purposes (import of metabolites into the matrix via symporters).
• Some heat production is essential (thermoregulation in endotherms).

Pyruvate is at a metabolic crossroads. Its fate depends on cellular conditions:

Aerobic conditions (with oxygen):

• Pyruvate enters the mitochondria and is converted to acetyl CoA by pyruvate dehydrogenase.
• Acetyl CoA enters the Krebs cycle.

Anaerobic conditions (without oxygen):

• In animals: pyruvate is converted to lactate by lactate dehydrogenase.
• In yeast: pyruvate is decarboxylated to ethanal and then reduced to ethanol by alcohol dehydrogenase.

Fatty acid oxidation (beta-oxidation):

• Fatty acids are broken down in the mitochondrial matrix by the removal of 2-carbon units as acetyl CoA.
• Each round of beta-oxidation produces 1 $\mathrm{FADH_2}$1 NADH, and 1 acetyl CoA.
• The acetyl CoA enters the Krebs cycle.
• Fatty acids yield more ATP per gram than carbohydrates (see Section 7.1).

Amino acid catabolism:

• Amino acids are deaminated (amino group removed as ammonia, converted to urea in the liver).
• The carbon skeleton is converted to intermediates of glycolysis or the Krebs cycle (pyruvate, acetyl CoA, $\alpha$-ketoglutarate, succinyl CoA, oxaloacetate).
• This explains the caloric value of proteins ($\approx 17\ \mathrm{kJ\ g^{-1}}$).

During intense exercise, lactate produced by anaerobic respiration in muscles diffuses into the bloodstream and is carried to the liver. In the liver, lactate is converted back to pyruvate, which is converted to glucose by gluconeogenesis (requiring ATP). The glucose is released back into the blood and taken up by muscles. This cycle is called the Cori cycle.

The net effect of the Cori cycle is to transport lactate from muscles to the liver for conversion back to glucose, at the cost of ATP in the liver. The oxygen debt accumulated during exercise must be “repaid” after exercise by continued elevated breathing to oxidise the lactate that accumulated.

Phosphofructokinase (PFK) is the key regulatory enzyme of glycolysis:

• Activated by: AMP (signals low ATP), fructose-2,6-bisphosphate (signals high glucose availability), ADP.
• Inhibited by: ATP (signals sufficient energy), citrate (signals abundant TCA cycle intermediates), low pH.

When ATP is abundant, PFK is inhibited, slowing glycolysis. When ATP is depleted (high AMP), PFK is activated, accelerating glycolysis. This ensures that glycolysis runs only when the cell needs more ATP.

Pyruvate dehydrogenase (link reaction) and isocitrate dehydrogenase and $\alpha$-ketoglutarate dehydrogenase (Krebs cycle) are also regulated by product inhibition and covalent modification (phosphorylation/dephosphorylation by kinases/phosphatases).

Using a respirometer with soda lime:

When soda lime is present, $\mathrm{CO_2}$ is absorbed, so the volume change measured represents only $\mathrm{O_2}$ consumption. This simplifies calculations because the only gas exchange occurring is $\mathrm{O_2}$ uptake.

Without soda lime:

If no $\mathrm{CO_2}$ absorbent is used, the volume change is the net difference between $\mathrm{O_2}$ consumed and $\mathrm{CO_2}$ produced. For carbohydrate substrates where $RQ = 1$This is zero (volumes cancel). For lipid substrates where $RQ < 1$The measured volume change underestimates $\mathrm{O_2}$ consumption.

$\text{True}\ \mathrm{O_2}\text{consumption} = \text{Measured volume change} \times \frac{1}{1 - RQ}$

A respirometer with soda lime measures an $\mathrm{O_2}$ consumption of $3.0\ \mathrm{cm^3}$ in 20 minutes. A separate respirometer without soda lime measures a net volume change of $1.5\ \mathrm{cm^3}$ in the same time.

From the soda lime respirometer: $\mathrm{O_2}$ consumed $= 3.0\ \mathrm{cm^3}$.

From the non-soda lime respirometer: net volume change $= \mathrm{O_2}$ consumed $- \mathrm{CO_2}$ produced $= 1.5\ \mathrm{cm^3}$.

Therefore: $\mathrm{CO_2}$ produced $= 3.0 - 1.5 = 1.5\ \mathrm{cm^3}$.

$RQ = \frac{1.5}{3.0} = 0.50$

An RQ of 0.50 suggests a mix of carbohydrate and lipid substrates being respired. A purely carbohydrate substrate would give $RQ = 1.0$; a purely lipid substrate would give $RQ \approx 0.7$. An RQ of 0.50 is lower than expected for either pure substrate, which may indicate the use of protein as a respiratory substrate (average protein $RQ \approx 0.8$) or experimental error.

• Evaporation of water from the respiring organism adds gas to the system, overestimating respiration rate.
• Temperature fluctuations cause thermal expansion/contraction of gases.
• Leaks in the apparatus reduce accuracy.
• Soda lime may become saturated (limited $\mathrm{CO_2}$ absorption capacity).
• The organism may not be at a steady state of respiration at the start of the experiment.

Worked Example: Palmitic acid ($\mathrm{C_{16}H_{32}O_2}$).

Palmitic acid undergoes 7 rounds of beta-oxidation, producing 7 acetyl CoA, 7 $\mathrm{FADH_2}$And 7 NADH:

• Beta-oxidation products: 7 $\mathrm{FADH_2} \times 1.5 = 10.5$ ATP; 7 NADH $\times 2.5 = 17.5$ ATP.
• 7 acetyl CoA enter the Krebs cycle: $7 \times$ (3 ATP from 3 NADH + 1 ATP from 1 $\mathrm{FADH_2}$ + 1 GTP) = $7 \times 5 = 35$ ATP.
• Activation cost: 2 ATP (to convert fatty acid to fatty acyl CoA in the cytoplasm).

Total ATP $= 10.5 + 17.5 + 35 - 2 = 61$ ATP.

Energy value: $61 \times 30.5 = 1860.5\ \mathrm{kJ\ mol^{-1}}$.

Compare with glucose: $32 \times 30.5 = 976\ \mathrm{kJ\ mol^{-1}}$.

Palmitic acid (256 $\mathrm{g\ mol^{-1}}$) produces approximately 1860\ \mathrm{kJ\ mol^{-1}Or $7.27\ \mathrm{kJ\ g^{-1}}$. Glucose (180 $\mathrm{g\ mol^{-1}}$) produces approximately $976\mathrm{kJ\ mol^{-1}}$Or $5.42\ \mathrm{kJ\ g^{-1}}$.

Fatty acids produce approximately 1.3 times more energy per gram than glucose, consistent with their higher energy value ($\approx 39\ \mathrm{kJ\ g^{-1}}$ vs $\approx 15.8\ \mathrm{kJ\ g^{-1}}$).

During intense exercise, the body’s $\mathrm{O_2}$ supply is insufficient to meet ATP demand. The body switches to anaerobic respiration, accumulating lactate. The oxygen debt is the volume of $\mathrm{O_2}$ required to oxidise the accumulated lactate after exercise.

Worked Example. A runner accumulates $500\ \mathrm{mg}$ of lactate during a sprint.

The oxidation of lactate:

$\mathrm{C_3H_6O_3 + 3O_2 \to 3CO_2 + 3H_2O}$

Molar mass of lactate $= 90\ \mathrm{g\ mol^{-1}}$.

Moles of lactate $= \frac{0.500}{90} = 0.00556\ \mathrm{mol}$.

$\mathrm{O_2}$ required $= 0.00556 \times 3 = 0.0167\ \mathrm{mol}$.

Volume of $\mathrm{O_2}$ at room temperature (assuming $1\ \mathrm{mol}$ gas $\approx 24\ \mathrm{dm^3}$):

$V = 0.0167 \times 24 = 0.40\ \mathrm{dm^3} = 400\ \mathrm{cm^3}$

The runner must breathe an additional $400\ \mathrm{cm^3}$ of $\mathrm{O_2}$ above resting requirements to fully repay the oxygen debt.

Tissues with high aerobic demand have more mitochondria per cell:

Mitochondria are thought to have originated from free-living aerobic bacteria that were engulfed by a larger anaerobic host cell approximately 2 billion years ago (the endosymbiosis theory, proposed by Lynn Margulis).

Evidence supporting endosymbiosis:

1. Stating that glycolysis produces “2 ATP” without specifying net yield. Glycolysis produces 4 ATP but uses 2 ATP, giving a net yield of 2 ATP. In examination answers, always specify the net yield. Also, glycolysis produces 2 molecules of pyruvate, 2 NADH, and 2 H₂O — not just ATP.

2. Confusing the roles of NADH and FADH₂. Both carry electrons to the ETC, but NADH donates at Complex I (pumping 10 H⁺ total) while FADH₂ donates at Complex II (pumping 6 H⁺ total). This means NADH generates approximately 2.5 ATP while FADH₂ generates approximately 1.5 ATP.

3. Stating that oxygen “makes ATP.” Oxygen is needed specifically as the final electron acceptor in the electron transport chain. The ATP itself is synthesised by ATP synthase, driven by the proton gradient. Oxygen’s role is to keep the electron transport chain flowing so that the proton gradient is maintained.

4. Confusing the Krebs cycle with the link reaction. The link reaction converts pyruvate to acetyl CoA (producing 1 NADH and 1 CO₂ per pyruvate). The Krebs cycle oxidises acetyl CoA completely (producing 3 NADH, 1 FADH₂, 1 GTP, and 2 CO₂ per turn). Both occur in the mitochondrial matrix.

5. Forgetting that anaerobic respiration is necessary to regenerate NAD⁺. The purpose of converting pyruvate to lactate (or ethanol) is not to produce energy but to regenerate NAD⁺ so that glycolysis can continue. Without NAD⁺, glycolysis stops at step 6 and no ATP is produced.

Cyanide binds with very high affinity to the ferric ($\mathrm{Fe^{3+}}$) state of cytochrome $a a_3$ (Complex IV), preventing the transfer of electrons to molecular oxygen. Without Complex IV function:

• $\mathrm{O_2}$ cannot be reduced to $\mathrm{H_2O}$.
• The entire ETC backs up (electrons cannot flow from Complex I or III).
• NADH and $\mathrm{FADH_2}$ cannot be oxidised, so the Krebs cycle and link reaction also stop (NAD$^+$ and FAD are not regenerated).
• ATP production ceases, and cells die rapidly. The brain and heart are most affected (highest $\mathrm{O_2}$ demand).

Treatment involves:

• Amyl nitrite or sodium nitrite: converts some haemoglobin to methaemoglobin ($\mathrm{Fe^{3+}}$), which has a higher affinity for cyanide than cytochrome oxidase. Cyanide dissociates from cytochrome oxidase and binds to methaemoglobin instead.
• Sodium thiosulfate: provides sulphur to convert cyanide to thiocyanate ($\mathrm{SCN^-}$), which is much less toxic and is excreted in urine.

Brown adipose tissue (BAT) in mammals contains large numbers of mitochondria with a unique protein called uncoupling protein 1 (UCP1, thermogenin). UCP1 forms a proton channel in the inner mitochondrial membrane, allowing $\mathrm{H^+}$ to flow back into the matrix without passing through ATP synthase.

The proton gradient is dissipated as heat instead of being used to make ATP. This is called non-shivering thermogenesis and is particularly important in newborn infants (who have limited ability to shiver) and in hibernating animals.

Newborns have more brown fat relative to body weight than adults. Brown fat is located around the neck, shoulders, and spine, where it can warm the blood supplying the brain and spinal cord.

Yeast (Saccharomyces cerevisiae) is a facultative anaerobe: it can respire aerobically or anaerobically.

Anaerobic respiration in yeast (alcoholic fermentation):

$\text{Glucose} \to 2\ \text{ethanol} + 2\ \mathrm{CO_2} + 2\ \text{ATP}$

This is used in:

• Bread making: $\mathrm{CO_2}$ produced causes the dough to rise (leavening). The ethanol evaporates during baking.
• Brewing: yeast ferments sugars in malted barley (or grapes) to produce ethanol. The process is carried out under anaerobic conditions to ensure ethanol production (if $\mathrm{O_2}$ is present, yeast will respire aerobically, producing $\mathrm{CO_2}$ and $\mathrm{H_2O}$ instead of ethanol, reducing the alcohol yield).

$\text{Glucose} \to 2\ \text{lactate} + 2\ \text{ATP}$

Lactate fermentation occurs in:

• Skeletal muscle during intense exercise (when $\mathrm{O_2}$ supply is insufficient for aerobic respiration).
• Red blood cells (which have no mitochondria and rely entirely on anaerobic glycolysis for ATP).
• Some bacteria (Lactobacillus) are used in the production of yoghurt, cheese, and sauerkraut.

Anaerobic respiration is extremely inefficient compared to aerobic respiration. However, it has the advantage of speed (glycolysis can produce ATP much faster than the full aerobic pathway) and does not require $\mathrm{O_2}$.

The link reaction converts pyruvate to acetyl CoA and is catalysed by the pyruvate dehydrogenase complex (PDC), a massive multi-enzyme complex containing three enzymes and five coenzymes:

Additional coenzyme: CoA (coenzyme A, derived from pantothenic acid, vitamin $\mathrm{B_5}$).

Overall: $\text{Pyruvate} + \text{CoA} + \text{NAD}^+ \to \text{acetyl CoA} + \mathrm{CO_2} + \text{NADH} + \text{H}^+$

Because several coenzymes are derived from vitamins, vitamin deficiencies impair respiration:

During intense exercise, the body’s $\mathrm{O_2}$ demand exceeds supply. The body switches to anaerobic respiration, producing lactate. The oxygen debt has two components:

1. Alactacid oxygen debt (fast component): the $\mathrm{O_2}$ needed to restore ATP and creatine phosphate stores, and to reload myoglobin with $\mathrm{O_2}$. This is repaid within minutes after exercise stops.

2. Lactacid oxygen debt (slow component): the $\mathrm{O_2}$ needed to oxidise accumulated lactate in the liver (via the Cori cycle). This takes hours to repay fully.

Worked Example. A runner completes a 400 m sprint in 50 seconds. During the sprint, their respiration rate was insufficient to meet ATP demand, so anaerobic respiration contributed significantly to ATP production.

If the runner’s peak $\mathrm{O_2}$ consumption during the sprint was $4.0\ \mathrm{L\ min^{-1}}$ and their resting $\mathrm{O_2}$ consumption is $0.25\ \mathrm{L\ min^{-1}}$:

Total $\mathrm{O_2}$ consumed during sprint $= (4.0 - 0.25) \times \frac{50}{60} = 3.75 \times 0.833 = 3.125\ \mathrm{L}$.

$\mathrm{O_2}$ deficit (oxygen that would have been consumed at peak rate if it had been maintained): this is difficult to calculate precisely, but if the total ATP demand during the sprint was met by aerobic respiration alone, the required $\mathrm{O_2}$ would have been approximately 8 L.

Estimated lactate production: $8 - 3.125 \approx 5\ \mathrm{L\ O_2}$ equivalent.

To oxidise 5 L of lactate (assuming 1 mole $\mathrm{O_2}$ per mole of lactate): this would require extended elevated breathing after the sprint to repay the lactacid debt.

Endurance training (e.g., marathon running) causes adaptations that improve aerobic capacity:

Sprint training (e.g., 100 m sprint) causes adaptations that improve anaerobic capacity:

$\dot{V}\mathrm{O_2}$ max (maximal oxygen uptake) is the maximum rate of $\mathrm{O_2}$ consumption during exercise. It is a measure of cardiorespiratory fitness.

Typical values:

$\dot{V}\mathrm{O_2}$ max is determined by the Fick equation:

$\dot{V}\mathrm{O_2\ max} = Q \times (C_a - C_v)$

Where $Q$ = cardiac output (L/min), $C_a$ = arterial $\mathrm{O_2}$ content, $C_v$ = venous $\mathrm{O_2}$ content. The difference $(C_a - C_v)$ is the arteriovenous $\mathrm{O_2}$ difference.

If cyanide is added to a suspension of mitochondria respiring on pyruvate:

If DNP is added to a suspension of mitochondria:

1. Cristae: the inner membrane is folded into cristae, greatly increasing the surface area for the ETC and ATP synthase. Cells with high energy demands (e.g., cardiac muscle cells, sperm cells) have mitochondria with more densely packed cristae.
2. Double membrane: the inner membrane is impermeable to $\mathrm{H^+}$ ions, allowing the maintenance of a proton gradient.
3. Own DNA and ribosomes: mitochondria can produce some of their own proteins (they code for 13 proteins involved in the ETC). Most mitochondrial proteins are encoded by nuclear DNA and imported.
4. Matrix enzymes: high concentration of enzymes for the Krebs cycle and link reaction.

Like chloroplasts, mitochondria are thought to have evolved from free-living aerobic bacteria engulfed by a larger host cell (endosymbiotic theory). Evidence:

1. Mitochondria have their own circular DNA (16.5 kbp in humans, encoding 13 proteins).
2. Mitochondria have 70S ribosomes (bacterial type, sensitive to chloramphenicol).
3. Mitochondria are surrounded by a double membrane.
4. Mitochondria reproduce by binary fission.
5. Mitochondrial DNA is maternally inherited (sperm mitochondria are destroyed after fertilisation).

Mutations in mitochondrial DNA (mtDNA) cause a range of diseases because mitochondria are essential for ATP production in all tissues:

Because mtDNA is maternally inherited, all children of an affected mother will inherit the mutation, but no children of an affected father will. The severity of mitochondrial diseases depends on heteroplasmy: the proportion of mutant vs normal mtDNA in each cell.

Location: mitochondrial matrix.

$\text{Pyruvate (3C)} + \mathrm{NAD^+} + \text{CoA} \to \text{acetyl CoA (2C)} + \mathrm{CO_2} + \mathrm{NADH}$

For each glucose molecule, the link reaction occurs twice (one for each pyruvate).

Key points:

• Pyruvate is decarboxylated (1 carbon removed as $\mathrm{CO_2}$).
• Pyruvate is dehydrogenated ($\mathrm{NAD^+}$ reduced to $\mathrm{NADH}$).
• The remaining 2-carbon fragment is attached to coenzyme A (CoA-SH) to form acetyl CoA.
• The enzyme complex is pyruvate dehydrogenase; it requires 5 coenzymes: $\mathrm{NAD^+}$FAD, CoA, thiamine pyrophosphate (TPP, derived from vitamin $\mathrm{B_1}$), and lipoic acid.

Location: mitochondrial matrix.

Per glucose (two turns of the cycle):

Yeast carries out alcoholic fermentation in the absence of $\mathrm{O_2}$:

$\text{Glucose} \to 2\text{ pyruvate} \to 2\text{ ethanol} + 2\mathrm{CO_2} + 2\text{ ATP}$

Steps:

1. Glycolysis produces 2 pyruvate, 2 ATP (net), and 2 NADH.
2. Pyruvate is decarboxylated by pyruvate decarboxylase to acetaldehyde (ethanal) + $\mathrm{CO_2}$.
3. Acetaldehyde is reduced to ethanol by alcohol dehydrogenase, regenerating $\mathrm{NAD^+}$ from NADH.

The regeneration of $\mathrm{NAD^+}$ is essential: without it, glycolysis would stop (no $\mathrm{NAD^+}$ available to accept electrons).

Applications: brewing (beer, wine); baking (the $\mathrm{CO_2}$ produced causes dough to rise); biofuel production (bioethanol).

Mammalian muscle cells carry out lactate fermentation during intense exercise:

$\text{Glucose} \to 2\text{ pyruvate} \to 2\text{ lactate} + 2\text{ ATP}$

Steps:

1. Glycolysis produces 2 pyruvate, 2 ATP (net), and 2 NADH.
2. Pyruvate is reduced to lactate by lactate dehydrogenase, regenerating $\mathrm{NAD^+}$ from NADH.

Lactate accumulates in the muscle and blood, lowering pH (causing fatigue and cramp). Lactate is transported to the liver via the blood and converted back to pyruvate (then glucose) by the Cori cycle.

Glycolysis occurs in the cytoplasm and does not require $\mathrm{O_2}$. It consists of two phases:

Phase 1: Energy investment (uses 2 ATP)

Phase 2: Energy payoff (produces 4 ATP + 2 NADH)

Net ATP yield from glycolysis: 4 - 2 = 2 ATP per glucose.

The three irreversible steps (1, 3, and 9) are the regulatory points:

Total $\mathrm{H^+}$ pumped per NADH: 4 (Complex I) + 4 (Complex III) + 2 (Complex IV) = 10 $\mathrm{H^+}$.

Total $\mathrm{H^+}$ pumped per $\mathrm{FADH_2}$: 0 (Complex II) + 4 (Complex III) + 2 (Complex IV) = 6 $\mathrm{H^+}$.

ATP synthase stoichiometry: approximately 4 $\mathrm{H^+}$ per ATP (3 $\mathrm{H^+}$ for ATP synthesis + 1 $\mathrm{H^+}$ for phosphate transport).

ATP yield per NADH: $10 / 4 = 2.5$ ATP.

ATP yield per $\mathrm{FADH_2}$: $6 / 4 = 1.5$ ATP.

Total ATP yield: approximately 30—32 ATP per glucose (depending on the shuttle used for cytoplasmic NADH).

                    ┌── Fatty acids ──→ β-oxidation ──→ acetyl CoA ──┐
                    │                                            │
Carbohydrates ──→ Glucose ──→ Glycolysis ──→ Pyruvate ──→ acetyl CoA ──┼── Krebs cycle ──→ CO2
                    │                                            │              │
Proteins ──→ Amino acids ──→ various entry points ────────────────┘              │
                                                                         │
                                                                    ETC ──→ ATP + H2O

The body can switch between metabolic fuels depending on circumstances:

During intense exercise, muscles may not receive enough $\mathrm{O_2}$ for aerobic respiration. Anaerobic respiration occurs:

$\text{Glucose} \to 2\ \text{lactate} + 2\ \text{ATP}$

The oxygen debt is the amount of extra $\mathrm{O_2}$ required after exercise to:

1. Oxidise the accumulated lactate back to pyruvate (which enters the Krebs cycle or is converted to glucose via gluconeogenesis).
2. Resynthesise ATP and phosphocreatine (PCr) stores.
3. Replace glycogen stores in the liver and muscles.

The Cori cycle describes the recycling of lactate between muscles and the liver: