AP · thinka-original Practice Paper

2024 AP AP Biology Practice Paper with Answers

Thinka May 2024 AP-Style Mock — AP Biology

34 marks90 mins2024

An original Thinka practice paper modelled on the structure and difficulty of the May 2024 AP AP Biology paper. Not affiliated with or reproduced from AP.

Section II - Part A: Long Free-Response Questions

Answer Question 1 and Question 2 in paragraph form. Allocate approximately 25 minutes per question. Standard mathematical formulas, chi-square tables, and reference sheets are provided.

2 Question · 18 marks

Question 1 · Long Free-Response

9 marks

Photosynthetic organisms convert light energy into chemical energy through light-dependent reactions in the thylakoid membranes of chloroplasts. During this process, electrons are extracted from water and transported through a series of protein complexes (Photosystem II, the cytochrome $b_6f$ complex, and Photosystem I) to reduce $\text{NADP}^+$ to $\text{NADPH}$, while simultaneously establishing a proton gradient used by ATP synthase to produce ATP.

Researchers are investigating the mechanism of action of a newly developed agricultural compound, Algicide-7 (A-7), on the photosynthetic electron transport chain of the unicellular green alga Chlorella vulgaris. Intact thylakoids were isolated from C. vulgaris cells and incubated in a buffered solution containing $\text{ADP}$, inorganic phosphate ($\text{P}_i$), and oxidized electron acceptors. Samples were exposed to constant illumination ($400\ \mu\text{mol photons}\cdot\text{m}^{-2}\cdot\text{s}^{-1}$) at $22^\circ\text{C}$ under various conditions. In Treatment 4, the artificial electron acceptor DCPIP was added; DCPIP accepts electrons directly from the primary electron acceptor of Photosystem II (PSII) before they reach the cytochrome $b_6f$ complex. Rates of oxygen evolution, ATP synthesis, and $\text{NADPH}$ production were measured and recorded in Table 1.

TABLE 1. RATES OF PHOTOSYNTHETIC ACTIVITY IN ISOLATED THYLAKOIDS UNDER VARYING EXPERIMENTAL TREATMENTS

| Treatment | Experimental Conditions | Rate of $\text{O}_2$ Evolution ($\mu\text{mol}\ \text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}\pm 2\text{SE}_x$) | Rate of ATP Synthesis ($\mu\text{mol ATP}\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}\pm 2\text{SE}_x$) | Rate of $\text{NADPH}$ Formation ($\mu\text{mol NADPH}\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}\pm 2\text{SE}_x$) |
| :--- | :--- | :--- | :--- | :--- |
| 1 | No A-7, $\text{NADP}^+$ present, Light | $142 \pm 6$ | $280 \pm 12$ | $138 \pm 5$ |
| 2 | $5\ \mu\text{M}$ A-7, $\text{NADP}^+$ present, Light | $71 \pm 4$ | $140 \pm 8$ | $69 \pm 4$ |
| 3 | $20\ \mu\text{M}$ A-7, $\text{NADP}^+$ present, Light | $8 \pm 2$ | $15 \pm 3$ | $7 \pm 2$ |
| 4 | $20\ \mu\text{M}$ A-7 + DCPIP, No $\text{NADP}^+$, Light | $140 \pm 7$ | $22 \pm 4$ | $0 \pm 0$ |
| 5 | No A-7, $\text{NADP}^+$ present, Dark | $0 \pm 0$ | $0 \pm 0$ | $0 \pm 0$ |

(a)
(i) Describe the biological function of water ($\text{H}_2\text{O}$) photolysis in the thylakoid lumen during the light-dependent reactions of photosynthesis.
(ii) Explain how the transfer of electrons along the photosynthetic electron transport chain establishes a proton concentration gradient across the thylakoid membrane.

(b)
(i) Identify the dependent variable measured to assess Photosystem II water-splitting activity in this experiment.
(ii) Identify the treatment that serves as a negative control to demonstrate that light energy is required for photosynthetic electron transport and ATP synthesis.

(c)
(i) Using the data in Table 1, calculate the percent decrease in the rate of ATP synthesis when isolated thylakoids are treated with $5\ \mu\text{M}$ A-7 compared to the untreated control (Treatment 1).
(ii) Based on Treatments 3 and 4, state the effect of adding DCPIP to thylakoids exposed to $20\ \mu\text{M}$ A-7 on the rate of $\text{O}_2$ evolution.
(iii) Based on the data in Table 1, justify the claim that A-7 inhibits electron flow downstream of Photosystem II (between PSII and PSI) rather than directly inhibiting the oxygen-evolving complex of Photosystem II.

(d)
(i) A researcher introduces an uncoupling reagent, FCCP, to untreated thylakoids in the light. FCCP creates channels in the thylakoid membrane that allow protons ($\text{H}^+$) to freely diffuse across the lipid bilayer. Predict the effect of adding FCCP on the rate of ATP synthesis AND on the rate of $\text{O}_2$ evolution compared to Treatment 1.
(ii) Explain the biological reasoning supporting your prediction regarding ATP synthesis.

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Worked solution

(a)(i)
* Photolysis of $\text{H}_2\text{O}$ splits water into electrons ($e^-$), protons ($\text{H}^+$), and molecular oxygen ($\text{O}_2$).
* The biological function is to supply replacement electrons to the oxidized reaction center chlorophyll $a$ ($\text{P680}^+$) in Photosystem II, enabling continuous light absorption and electron flow.

(a)(ii)
* High-energy electrons moving through the electron transport chain (specifically via plastoquinone and the cytochrome $b_6f$ complex) release free energy, which is used to actively pump protons ($\text{H}^+$) from the stroma across the thylakoid membrane into the thylakoid lumen.
* Combined with the release of protons from water photolysis in the lumen and the consumption of protons in the stroma during $\text{NADP}^+$ reduction, a steep transmembrane electrochemical/proton gradient (lower pH in lumen, higher pH in stroma) is generated.

(b)(i)
* Dependent variable: Rate of $\text{O}_2$ evolution / production ($\mu\text{mol}\ \text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$).

(b)(ii)
* Treatment 5 (no A-7, $\text{NADP}^+$ present, dark condition).

(c)(i)
* Calculation of percent decrease:
$$\text{Percent decrease} = \frac{\text{Initial Value} - \text{New Value}}{\text{Initial Value}} \times 100\%$$
$$\text{Percent decrease} = \frac{280 - 140}{280} \times 100\% = \frac{140}{280} \times 100\% = 50\%$$ (or $50.0\%$).

(c)(ii)
* The addition of DCPIP increases / restores the rate of $\text{O}_2$ evolution from $8 \pm 2$ to $140 \pm 7\ \mu\text{mol}\ \text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$ (a rate statistically indistinguishable from untreated controls).

(c)(iii)
* In Treatment 3, $20\ \mu\text{M}$ A-7 drastically reduces $\text{O}_2$ evolution because the entire electron transport chain becomes back-logged/blocked.
* However, in Treatment 4, when DCPIP is added to accept electrons directly from PSII, $\text{O}_2$ evolution is completely restored to control levels ($140$ vs $142\ \mu\text{mol}\ \text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$). This confirms that the oxygen-evolving complex and PSII reaction center are intact and functional, proving that A-7 blocks electron transport downstream of PSII (e.g., at the plastoquinone-binding site or cytochrome $b_6f$ complex).

(d)(i)
* Prediction for ATP synthesis: The rate of ATP synthesis will decrease / drop to near zero.
* Prediction for $\text{O}_2$ evolution: The rate of $\text{O}_2$ evolution will remain unchanged or increase (due to relief of photosynthetic control).

(d)(ii)
* ATP synthase requires a proton-motive force (electrochemical gradient of $\text{H}^+$) across the thylakoid membrane to catalyze the phosphorylation of $\text{ADP} + \text{P}_i \rightarrow \text{ATP}$.
* FCCP increases membrane permeability to $\text{H}^+$, allowing protons to freely diffuse back into the stroma without passing through ATP synthase, thereby dissipating the gradient needed to drive ATP synthesis.

Marking scheme

Part (a): 2 points maximum
* (a)(i) Description (1 point):
* Accept one of the following:
* Replaces electrons lost/donated by the reaction center chlorophyll (P680) of Photosystem II.
* Supplies electrons to Photosystem II and releases protons ($\text{H}^+$) into the lumen / releases oxygen ($\text{O}_2$) as a byproduct.
* (a)(ii) Explanation (1 point):
* Accept one of the following:
* Exergonic/redox reactions along the electron transport chain provide energy to pump protons ($\text{H}^+$) from the stroma into the thylakoid lumen.
* Proton accumulation in the thylakoid lumen generated by active proton pumping (cytochrome $b_6f$ / plastoquinone) and lumenal water oxidation creates a higher concentration of $\text{H}^+$ inside the lumen relative to the stroma.

Part (b): 2 points maximum
* (b)(i) Identification (1 point):
* Rate of oxygen ($\text{O}_2$) evolution/production.
* (b)(ii) Identification (1 point):
* Treatment 5 / the thylakoids incubated in the dark (with $\text{NADP}^+$ and no A-7).

Part (c): 3 points maximum
* (c)(i) Calculation (1 point):
* $50\%$ (or $50$, $0.50$; setup: $\frac{280 - 140}{280} \times 100$).
* (c)(ii) State (1 point):
* DCPIP increases / restores the rate of $\text{O}_2$ evolution (from $8$ to $140\ \mu\text{mol}\ \text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$).
* (c)(iii) Justification (1 point):
* Accept one of the following:
* In Treatment 4, providing an electron acceptor (DCPIP) directly after PSII completely restores $\text{O}_2$ production to control levels, indicating that PSII/water photolysis is fully functional and the block is downstream of PSII.
* If A-7 directly inhibited PSII/water splitting, adding an electron acceptor downstream of PSII (DCPIP) would not restore $\text{O}_2$ evolution.

Part (d): 2 points maximum
* (d)(i) Prediction (1 point):
* ATP synthesis will decrease / cease AND $\text{O}_2$ evolution will remain unchanged / increase / persist.
(Both predictions required for the 1 point.)
* (d)(ii) Explanation (1 point):
* Accept one of the following:
* FCCP dissipates/eliminates the proton ($\text{H}^+$) gradient (proton-motive force), which is required to drive rotation/catalytic activity of ATP synthase.
* Protons leak across the membrane instead of flowing through ATP synthase, preventing phosphorylation of ADP.

Question 2 · Long Free-Response

9 marks

Photosynthetic microalgae utilize dissolved inorganic carbon for the light-independent reactions of photosynthesis. In aquatic environments, inorganic carbon exists primarily as bicarbonate ions ($\text{HCO}_3^-$), which are transported into algal cells and converted into carbon dioxide ($\text{CO}_2$) near the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

Researchers cultured the unicellular green alga Isochrysis galbana under controlled conditions to evaluate how light intensity and bicarbonate concentration affect photosynthetic activity. Cultures were suspended in sealed, temperature-regulated chambers at $22^\circ\text{C}$. The rate of oxygen evolution was monitored over a 30-minute period across five bicarbonate concentrations ($0.0\text{ mM}$, $0.5\text{ mM}$, $1.0\text{ mM}$, $2.5\text{ mM}$, and $5.0\text{ mM}$) under either Low Light ($50\ \mu\text{mol photons}\cdot\text{m}^{-2}\cdot\text{s}^{-1}$) or High Light ($300\ \mu\text{mol photons}\cdot\text{m}^{-2}\cdot\text{s}^{-1}$). The results are summarized in Table 1.

**TABLE 1. MEAN RATE OF OXYGEN EVOLUTION IN Isochrysis galbana AT VARYING BICARBONATE CONCENTRATIONS UNDER LOW AND HIGH LIGHT INTENSITIES**

| Bicarbonate Concentration ($\text{mM}$) | Mean Rate of $\text{O}_2$ Evolution Under Low Light ($\mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1} \pm 2\text{SE}_\bar{x}$) | Mean Rate of $\text{O}_2$ Evolution Under High Light ($\mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1} \pm 2\text{SE}_\bar{x}$) |
| :---: | :---: | :---: |
| $0.0$ | $4.2 \pm 1.1$ | $5.1 \pm 1.2$ |
| $0.5$ | $14.5 \pm 1.8$ | $26.3 \pm 2.4$ |
| $1.0$ | $22.0 \pm 2.0$ | $45.8 \pm 3.1$ |
| $2.5$ | $28.4 \pm 2.2$ | $68.2 \pm 3.5$ |
| $5.0$ | $29.1 \pm 2.5$ | $70.0 \pm 3.8$ |

(a) Describe the biochemical process that directly generates oxygen gas ($\text{O}_2$) during photosynthesis.
Explain how the proton gradient established during the light-dependent reactions drives the production of ATP.

(b) Construct a line graph on the provided axes to represent the data in Table 1. On your graph, plot the mean rate of oxygen evolution under both Low Light and High Light conditions across the bicarbonate concentrations tested. Include error bars representing $\pm 2\text{SE}_\bar{x}$ for each data point and clearly label each line.

(c) Based on the data in Table 1, identify the bicarbonate concentration above which further increases in inorganic carbon do not result in a statistically significant increase in the rate of oxygen evolution under Low Light conditions.
Calculate the difference in the mean rate of oxygen evolution between High Light and Low Light conditions at a bicarbonate concentration of $2.5\text{ mM}$.

(d) A researcher introduces DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)—an herbicide that binds specifically to the plastoquinone-binding site of Photosystem II (PSII) and inhibits electron transfer—to a culture of I. galbana maintained at $5.0\text{ mM}$ bicarbonate and High Light.
Predict the effect of DCMU addition on the rate of oxygen evolution in the culture.
Justify your prediction based on the molecular mechanism of Photosystem II.

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Worked solution

Part (a)
* Describe: The splitting/photolysis of water molecules ($2\text{H}_2\text{O} \rightarrow \text{O}_2 + 4\text{H}^+ + 4e^-$) at the oxygen-evolving complex of Photosystem II (PSII) generates oxygen gas ($\text{O}_2$).
* Explain: As electrons move through the photosynthetic electron transport chain, protons ($\text{H}^+$) are pumped from the stroma into the thylakoid lumen (supplemented by protons released from water photolysis), establishing an electrochemical proton gradient. Protons diffuse down this electrochemical gradient from the thylakoid lumen back into the stroma through ATP synthase, causing conformational rotation of the enzyme that catalyzes the phosphorylation of ADP and inorganic phosphate ($\text{P}_i$) to form ATP (chemiosmosis/photophosphorylation).

Part (b)
* Graph Construction (3 points):
1. Axes and scale: Independent variable (Bicarbonate Concentration in $\text{mM}$) on the x-axis; dependent variable (Mean Rate of $\text{O}_2$ Evolution in $\mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$) on the y-axis. Scales are linear, proportional, and fully utilize at least 50% of the graphing space.
2. Plotting and Key: All 10 data points correctly plotted; lines connected between points or best fit per series; series clearly identified with a key or direct text labels for "Low Light" and "High Light".
3. Error Bars: Error bars representing $\pm 2\text{SE}_\bar{x}$ are accurately drawn for each data point according to Table 1 values.

Part (c)
* Identify: $2.5\text{ mM}$ (Accept: from $2.5\text{ mM}$ to $5.0\text{ mM}$). The error bars for $2.5\text{ mM}$ ($28.4 \pm 2.2$, range: $26.2$–$30.6$) and $5.0\text{ mM}$ ($29.1 \pm 2.5$, range: $26.6$–$31.6$) overlap substantially, indicating no statistically significant difference.
* Calculate:
$$\text{Difference} = 68.2 - 28.4 = 39.8\ \mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$$

Part (d)
* Predict: The rate of oxygen evolution will decrease (or drop to near zero / cease entirely).
* Justify: When DCMU binds to the plastoquinone-binding site of PSII, it blocks electron transport from the reaction center ($\text{P680}$) to downstream electron carriers (plastoquinone/cytochrome $b_6f$). Consequently, the oxidized reaction center ($\text{P680}^+$) cannot continue to accept electrons from the oxygen-evolving complex, halting the oxidation/photolysis of water into $\text{O}_2$.

Marking scheme

Part (a) (2 points maximum):
* 1 point for describing that oxygen is produced by the splitting / photolysis / oxidation of water ($\text{H}_2\text{O}$) at Photosystem II (PSII).
* 1 point for explaining that protons flow down their concentration/electrochemical gradient from the thylakoid lumen into the stroma through ATP synthase, driving the phosphorylation of ADP to ATP (chemiosmosis).

Part (b) (3 points maximum):
* 1 point for correct orientation, labeling, and linear scaling of both axes with units (x-axis: Bicarbonate Concentration / $\text{mM}$; y-axis: Mean Rate of $\text{O}_2$ Evolution / $\mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$).
* 1 point for correctly plotting all data points, connecting points with lines, and identifying each treatment with a distinct key or line label.
* 1 point for accurately plotting all error bars representing $\pm 2\text{SE}_\bar{x}$ on all 10 data points.

Part (c) (2 points maximum):
* 1 point for identifying $2.5\text{ mM}$ (or between $2.5\text{ mM}$ and $5.0\text{ mM}$) as the concentration where error bars overlap.
* 1 point for calculating the difference: $39.8\ \mu\text{mol }\text{O}_2\cdot\text{mg Chl}^{-1}\cdot\text{hr}^{-1}$ (Setup: $68.2 - 28.4$).

Part (d) (2 points maximum):
* 1 point for predicting that the rate of oxygen evolution will decrease / cease / approach zero.
* 1 point for justifying that blocking electron flow downstream of PSII prevents the re-oxidation of the reaction center ($\text{P680}$), thereby stopping the photolysis/splitting of water needed to supply replacement electrons.

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Section II - Part B: Short Free-Response Questions

Answer Questions 3 through 6 in paragraph form. Allocate approximately 10 minutes per question.

6 Question · 24 marks

Question 1 · free-response

4 marks

Researchers are investigating the regulation of cellular respiration in isolated mitochondria from spinach (Spinacia oleracea) leaves. The researchers suspend functional, intact mitochondria in an isotonic buffer containing pyruvate, ADP, and inorganic phosphate ($\text{P}_\text{i}$) at $25^\circ\text{C}$. The rate of oxygen ($\text{O}_2$) consumption is monitored over time using a dissolved oxygen probe.

After establishing a steady baseline rate of oxygen consumption, the researchers add Compound X to the suspension. Compound X is a synthetic chemical that specifically binds to and inhibits cytochrome c oxidase (Complex IV) of the mitochondrial electron transport chain.

(a) Describe the role of oxygen ($\text{O}_2$) in the electron transport chain during aerobic cellular respiration.

(b) Identify the dependent variable in the experiment.

(c) Predict the effect of adding Compound X to the mitochondrial suspension on the rate of ATP synthesis by ATP synthase.

(d) Justify your prediction in part (c) based on the relationship between the electron transport chain and chemiosmosis.

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Worked solution

(a) Oxygen functions as the final electron acceptor in the mitochondrial electron transport chain. It accepts low-energy electrons from Complex IV and combines with free protons ($\text{H}^+$) in the mitochondrial matrix to form water ($\text{H}_2\text{O}$), allowing the electron transport chain to maintain continuous electron flow.

(b) The dependent variable is the rate of oxygen ($\text{O}_2$) consumption (or the concentration of dissolved oxygen over time).

(c) The rate of ATP synthesis by ATP synthase will decrease (or cease entirely).

(d) When Compound X inhibits Complex IV, electron transport through the chain is halted, which stops the active pumping of protons ($\text{H}^+$) across the inner mitochondrial membrane into the intermembrane space. As a result, the proton concentration gradient / proton-motive force dissipates, so protons can no longer flow down their electrochemical gradient through ATP synthase to power the phosphorylation of ADP to ATP.

Marking scheme

Part (a): 1 point
- Describe the role of oxygen:
- Accept: (Oxygen acts as the) final / terminal electron acceptor (of the electron transport chain) / combines with electrons and protons ($\text{H}^+$) to form water ($\text{H}_2\text{O}$).

Part (b): 1 point
- Identify the dependent variable:
- Accept: (Rate of) oxygen / $\text{O}_2$ consumption / (change in) dissolved oxygen concentration (over time).

Part (c): 1 point
- Predict the effect of Compound X:
- Accept: (The rate of ATP synthesis will) decrease / stop / cease / be inhibited.

Part (d): 1 point
- Justify the prediction:
- Accept one of the following:
- Inhibiting Complex IV halts electron flow, which prevents/decreases proton ($\text{H}^+$) pumping into the intermembrane space, thereby reducing/eliminating the proton gradient / proton-motive force needed to drive ATP synthase.
- Without active electron transport, the electrochemical / proton gradient across the inner mitochondrial membrane is not maintained/generated, so chemiosmosis / proton flow through ATP synthase cannot occur.

Question 2 · free-response

4 marks

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Worked solution

Marking scheme

Question 3 · Conceptual Analysis

4 marks

Brown adipose tissue (BAT) plays a vital role in nonshivering thermogenesis in mammals. During cold exposure, sympathetic nervous system signaling stimulates intracellular lipolysis, producing free fatty acids that activate uncoupling protein 1 (UCP1). UCP1 is a transport channel embedded in the inner mitochondrial membrane that allows protons ($\text{H}^+$) to flow from the intermembrane space into the mitochondrial matrix.

(a) Describe the process by which the mitochondrial electron transport chain establishes the proton concentration gradient across the inner mitochondrial membrane.

(b) Explain how active UCP1 in the inner mitochondrial membrane leads to increased heat production instead of ATP synthesis.

(c) A researcher exposes a culture of brown adipocytes to a compound that specifically inhibits Complex IV (cytochrome c oxidase) of the electron transport chain. Predict the effect of this inhibitor on the rate of oxygen ($\text{O}_2$) consumption by the adipocytes.

(d) Justify your prediction in part (c).

Show answer & marking scheme

Worked solution

(a) As high-energy electrons from $\text{NADH}$ and $\text{FADH}_2$ are transferred through the protein complexes of the electron transport chain in a series of exergonic redox reactions, the released free energy is used to pump protons ($\text{H}^+$) against their concentration gradient from the mitochondrial matrix into the intermembrane space, creating an electrochemical proton gradient.

(b) Under normal conditions, protons re-enter the mitochondrial matrix through ATP synthase, coupling the exergonic flow of protons to the phosphorylation of ADP to generate ATP (chemiosmosis). UCP1 creates an alternative, uncoupled pathway that allows protons to diffuse down their electrochemical gradient directly into the matrix without passing through ATP synthase. Because the proton motive force is dissipated without performing chemical work (ATP synthesis), the stored potential energy of the electrochemical gradient is released directly as thermal energy (heat).

(c) The rate of oxygen ($\text{O}_2$) consumption will decrease (or stop entirely).

(d) Complex IV (cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain responsible for catalyzing the reduction of molecular oxygen (the terminal electron acceptor) to water. When Complex IV is inhibited, electron transfer to oxygen cannot occur, which halts the consumption of $\text{O}_2$.

Marking scheme

Part (a): 1 point
- Describe the mechanism of gradient formation.
* Accept one of the following:
- Energy released from the (exergonic) transfer of electrons through the electron transport chain / redox reactions is used to pump/transport protons ($\text{H}^+$) from the (mitochondrial) matrix into the intermembrane space.
- The sequential oxidation-reduction reactions along electron transport chain complexes actively pump $\text{H}^+$ across the inner membrane against their gradient.

Part (b): 1 point
- Explain how active UCP1 generates heat rather than ATP.
* Accept one of the following:
- UCP1 allows protons ($\text{H}^+$) to flow down their electrochemical gradient into the matrix bypassing ATP synthase, so the potential energy stored in the gradient is dissipated/released as heat / thermal energy rather than being captured as ATP / chemical energy.
- UCP1 uncouples the proton gradient from ATP synthase / phosphorylation of ADP, converting the proton motive force directly into heat.

Part (c): 1 point
- Predict the effect of Complex IV inhibition.
* Accept one of the following:
- Oxygen ($\text{O}_2$) consumption will decrease / decline / stop / cease.

Part (d): 1 point
- Justify the prediction.
* Accept one of the following:
- Complex IV is responsible for transferring electrons to oxygen / $\text{O}_2$ (the terminal / final electron acceptor); inhibiting Complex IV prevents electron transfer to oxygen, halting oxygen reduction / consumption.
- Oxygen is reduced (to water) at Complex IV; blocking this complex prevents oxygen from acting as the final electron acceptor.

Question 4 · Analyze Model or Visual Representation of a Biological Concept or Process

4 marks

Acid hydrolases are hydrolytic enzymes that function exclusively within lysosomes to degrade macromolecules. Figure 1 illustrates the intracellular trafficking pathway responsible for delivering newly synthesized acid hydrolases from their site of translation to the lysosomal lumen.

```
Figure 1
Trafficking Pathway of Lysosomal Acid Hydrolases

[ Ribosome + RER ]
│ (Synthesis & translocation into lumen)
▼
[ cis-Golgi ] ────► Phosphotransferase adds Mannose-6-Phosphate (M6P) tag
│
▼
[ trans-Golgi ] ───► M6P Receptors (MPRs) bind M6P-tagged hydrolases
│ and package them into clathrin-coated vesicles
▼
[ Transport Vesicle ]
│
▼
[ Late Endosome / ] ─► Low pH (pH ≈ 5.0) triggers dissociation of hydrolase
[ Lysosome ] from MPR; active hydrolase degrades substrates.
│
└───────────► Free MPR is recycled back to trans-Golgi
```

(a) Based on the model, describe the role of the mannose-6-phosphate (M6P) tag in determining the intracellular destination of the acid hydrolase.

(b) Explain why acid hydrolases that accidentally leak into the cytosol ($\text{pH} \approx 7.2$) do not cause extensive catalytic damage to cytoplasmic components.

(c) A genetic mutation results in a completely nonfunctional phosphotransferase enzyme in the cis-Golgi. Predict the primary location where newly synthesized acid hydrolases will be found in individuals with this mutation.

(d) Justify your prediction in part (c) using the trafficking model provided.

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Worked solution

(a) The mannose-6-phosphate (M6P) tag serves as a sorting/targeting signal that is recognized and bound by specific M6P receptors (MPRs) in the trans-Golgi network, ensuring that the enzyme is packaged into vesicles destined for the endosomal/lysosomal system rather than entering the constitutive secretory pathway.

(b) Lysosomal enzymes are acid hydrolases designed to function at a low, acidic pH ($\approx 5.0$). The neutral/slightly alkaline pH of the cytosol ($\approx 7.2$) alters the protonation of active-site residues or the tertiary conformation of the enzyme, rendering the hydrolases largely inactive and preventing non-specific degradation of cytosolic organelles and macromolecules.

(c) The acid hydrolases will be secreted into the extracellular fluid / outside the cell (or found in the extracellular medium).

(d) In the absence of functional phosphotransferase activity, the M6P tag cannot be added to the acid hydrolases in the cis-Golgi. Consequently, the hydrolases cannot bind to M6P receptors in the trans-Golgi network for diversion to lysosomes, and the default secretory pathway carries them via secretory vesicles to the plasma membrane where they are exocytosed.

Marking scheme

(a) Describe the role of the M6P tag (1 point)
* Accept one of the following:
* It serves as a molecular sorting/targeting signal that binds to M6P receptors in the trans-Golgi to direct packaging into vesicles targeted to lysosomes/endosomes.
* It prevents the acid hydrolase from following the default secretory pathway by enabling recognition and sorting by membrane-bound receptors in the Golgi.

(b) Explain why leaked hydrolases do not cause extensive cytosolic damage (1 point)
* Accept one of the following:
* Acid hydrolases require an acidic pH ($\text{pH} \approx 5.0$) for optimal activity; the higher/neutral pH of the cytosol ($\text{pH} \approx 7.2$) inactivates/denatures the enzymes / reduces their catalytic rate.
* The neutral pH of the cytosol alters the tertiary structure / active site conformation of the acid hydrolases, preventing them from effectively binding or hydrolyzing cytoplasmic substrates.

(c) Predict the location of acid hydrolases (1 point)
* Accept one of the following:
* In the extracellular fluid / space / outside the cell / secreted into the bloodstream or culture medium.

(d) Justify the prediction (1 point)
* Accept one of the following:
* Without the M6P tag, the enzymes cannot bind to the M6P receptors (in the trans-Golgi), causing them to be packaged into default secretory vesicles that fuse with the plasma membrane / are exocytosed.
* Lysosomal targeting requires receptor-ligand binding between M6P and MPR; without the modification, the proteins enter the constitutive bulk exocytosis/secretory pathway.

Question 5 · Analyze Model or Visual Representation of a Biological Concept or Process

4 marks

Figure 1 shows a model of the light-dependent reactions of photosynthesis occurring across the thylakoid membrane in a plant chloroplast.

```
Figure 1
Model of Chloroplast Thylakoid Membrane Electron Transport

STROMA (pH ≈ 8.0)
─────────────────────────────────────────────────────────────────────────
Thylakoid [ PS II ] ──► [ Plastoquinone / ] ──► [ PS I ] ──► [ Ferredoxin / ] ──► NADPH
Membrane [ Cytochrome b6f ] [ NADP+ Red. ]
▲ │
│ ▼ (H+ pumped)
H2O ──┴──► O2 + H+ (H+)
─────────────────────────────────────────────────────────────────────────
THYLAKOID LUMEN (pH ≈ 5.0) ──────────────► [ ATP Synthase ] ──► ATP
(H+ flow to stroma)
```

(a) Based on the model, identify the primary source of electrons that replenishes the reaction center chlorophyll molecules of Photosystem II (PS II).

(b) Describe the structural feature of the thylakoid membrane that allows the establishment and maintenance of the proton ($\text{H}^+$) concentration gradient shown in Figure 1.

(c) A researcher treats isolated, illuminated chloroplasts with a chemical uncoupler that makes the thylakoid membrane fully permeable to protons ($\text{H}^+$). Predict the effect of this treatment on the rate of ATP synthesis by ATP synthase.

(d) Justify your prediction in part (c) based on the mechanism of ATP synthase shown in the model.

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Worked solution

(a) The primary source of electrons is water ($\text{H}_2\text{O}$), which undergoes photolysis/splitting at Photosystem II to release electrons, protons, and oxygen gas.

(b) The thylakoid membrane possesses a phospholipid bilayer with a hydrophobic interior, rendering it impermeable to hydrophilic/charged particles such as hydrogen ions ($\text{H}^+$). This barrier prevents spontaneous leakage of protons back into the stroma, thereby maintaining compartmentalization and a distinct electrochemical proton gradient.

(c) ATP synthesis will significantly decrease or cease completely.

(d) ATP synthase requires a proton-motive force (electrochemical gradient of $\text{H}^+$) across the thylakoid membrane, where protons flow down their concentration gradient from the lumen to the stroma through the enzyme. The uncoupler allows protons to equilibrate across the membrane, eliminating the gradient and the kinetic energy needed to rotate ATP synthase and catalyze the formation of ATP from ADP and inorganic phosphate.

Marking scheme

(a) Identify the electron source (1 point)
* Accept one of the following:
* Water / $\text{H}_2\text{O}$
* Splitting / photolysis of water

(b) Describe the structural feature (1 point)
* Accept one of the following:
* The hydrophobic core / interior of the phospholipid bilayer is impermeable to ions/protons ($\text{H}^+$).
* It is a closed, semipermeable / selectively permeable membrane that separates the lumen from the stroma, preventing free diffusion of charged protons.

(c) Predict the effect on ATP synthesis rate (1 point)
* Accept one of the following:
* ATP synthesis will decrease / stop / cease.

(d) Justify the prediction (1 point)
* Accept one of the following:
* The chemical uncoupler dissipates / eliminates the proton ($\text{H}^+$) gradient (proton-motive force), which is required to drive the mechanical rotation / catalytic activity of ATP synthase as protons flow from the lumen to the stroma.
* Protons bypass ATP synthase by moving freely across the membrane, so there is no proton flow through ATP synthase to drive the phosphorylation of $\text{ADP}$ to $\text{ATP}$.

Question 6 · Analyze Data

4 marks

Researchers investigated the effect of a synthetic chemical, Compound X, on the metabolic activity of isolated plant mitochondria. Suspensions of intact, isolated mitochondria were incubated in a buffered solution containing pyruvate, excess $\text{ADP}$, and inorganic phosphate ($\text{P}_i$). Different concentrations of Compound X were added to the suspensions, and the rates of oxygen ($\text{O}_2$) consumption and $\text{ATP}$ synthesis were measured over a 10-minute period. The results are shown in Table 1.

**TABLE 1. RATE OF $\text{O}_2$ CONSUMPTION AND $\text{ATP}$ SYNTHESIS IN ISOLATED MITOCHONDRIA TREATED WITH COMPOUND X**

| Concentration of Compound X ($\mu\text{M}$) | Rate of $\text{O}_2$ Consumption ($\text{nmol } \text{O}_2\text{/min}\cdot\text{mg protein}$) (Mean $\pm 2\text{SE}_{\bar{x}}$) | Rate of $\text{ATP}$ Synthesis ($\text{nmol ATP/min}\cdot\text{mg protein}$) (Mean $\pm 2\text{SE}_{\bar{x}}$) |
| :--- | :--- | :--- |
| 0 (No Compound X) | $142 \pm 6$ | $426 \pm 18$ |
| 5 | $139 \pm 8$ | $280 \pm 14$ |
| 20 | $145 \pm 7$ | $112 \pm 12$ |
| 50 | $141 \pm 6$ | $14 \pm 4$ |

(a) Identify the control treatment in this experiment.

(b) Based on the data in Table 1, describe the relationship between the concentration of Compound X and the rate of $\text{ATP}$ synthesis.

(c) Based on the data in Table 1, explain whether Compound X acts by directly inhibiting the protein complexes of the electron transport chain.

(d) In a functioning mitochondrion, protons ($\text{H}^+$) are actively pumped across the inner membrane. If Compound X functions as an uncoupling agent that makes the inner mitochondrial membrane permeable to protons, predict the effect of $50\ \mu\text{M}$ Compound X on the pH of the intermembrane space compared to the control.

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Worked solution

(a) The control treatment is the incubation containing $0\ \mu\text{M}$ Compound X (mitochondria with pyruvate, ADP, and $\text{P}_i$ but no inhibitor added).

(b) As the concentration of Compound X increases from $0\ \mu\text{M}$ to $50\ \mu\text{M}$, the rate of $\text{ATP}$ synthesis decreases significantly (from $426$ to $14\ \text{nmol ATP/min}\cdot\text{mg protein}$).

(c) Compound X does not inhibit the electron transport chain. If the electron transport chain were inhibited, electrons would not flow to oxygen (the terminal electron acceptor), causing oxygen consumption to decrease. However, the data show that the rate of $\text{O}_2$ consumption remains statistically unchanged across all concentrations tested (all values fall within the margin of error $\pm 2\text{SE}_{\bar{x}}$).

(d) The pH of the intermembrane space will increase (become less acidic/higher pH). When protons leak back across the inner membrane into the matrix through the uncoupling pathway, the concentration of $\text{H}^+$ in the intermembrane space decreases, resulting in a higher pH.

Marking scheme

Part (a): 1 point
- Identify the control treatment:
* Accept: The $0\ \mu\text{M}$ Compound X treatment / the treatment with no Compound X added.

Part (b): 1 point
- Describe the relationship between Compound X concentration and ATP synthesis:
* Accept: An increase in the concentration of Compound X leads to a decrease / reduction in the rate of $\text{ATP}$ synthesis (or inverse/negative relationship).

Part (c): 1 point
- Explain whether Compound X directly inhibits the electron transport chain:
* Accept: It does not inhibit the electron transport chain because the rate of oxygen consumption does not change / remains constant across all concentrations (the error bars overlap), which indicates that electron transport to oxygen continues unimpeded.

Part (d): 1 point
- Predict the effect of Compound X on the pH of the intermembrane space:
* Accept: The pH will increase / become less acidic / become more basic (due to protons leaking back across the membrane into the matrix, reducing the $[\text{H}^+]$ in the intermembrane space).

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