Photosynthesis

The chloroplast has an internal membrane system of thylakoids stacked into grana. These membranes provide a large surface area that holds many photosystems and pigment molecules, so a large amount of light can be absorbed for the light-dependent reactions.

The thylakoid space (lumen) is a small enclosed compartment, so the H⁺ gradient built up during the light reactions becomes steep, which is needed for chemiosmotic ATP synthesis.

The stroma is the fluid surrounding the thylakoids and contains the enzymes of the Calvin cycle (including rubisco), so it is the site of the light-independent reactions.

Photolysis is the splitting of water using light energy within photosystem II: $2H_2O \rightarrow 4H^+ + 4e^- + O_2$.

The electrons released replace the electrons lost from the reaction-centre chlorophyll of photosystem II when it was excited, allowing the light reactions to continue.

The protons (H⁺) released contribute to the proton gradient and are later used, with electrons from photosystem I, to reduce NADP to reduced NADP. The oxygen produced is released as a by-product (the source of atmospheric oxygen).

In the stroma of the chloroplast, carbon dioxide combines with the 5-carbon acceptor molecule ribulose bisphosphate (RuBP).

This reaction is catalysed by the enzyme rubisco (ribulose bisphosphate carboxylase).

The unstable 6-carbon intermediate formed immediately breaks down into two molecules of glycerate-3-phosphate (GP), each containing 3 carbons.

GP is then reduced to triose phosphate (GALP) in a later step of the cycle, but the fixation step itself is the addition of CO₂ to RuBP by rubisco to make 2 GP.

Light energy is absorbed by the pigments of the photosystems in the thylakoid membrane, exciting electrons in the reaction-centre chlorophyll to a higher energy level so that they are emitted and taken up by an electron acceptor (the chlorophyll is photoionised). The electrons lost from photosystem II are replaced by the photolysis of water ($2H_2O \rightarrow 4H^+ + 4e^- + O_2$).

The excited electrons are passed along a chain of electron carriers (the electron transport chain) in the thylakoid membrane. As the electrons move down the chain they release energy, and this energy is used to pump protons (H⁺) from the stroma into the thylakoid space.

This creates a high concentration of H⁺ in the thylakoid space — an electrochemical (proton) gradient across the thylakoid membrane.

The protons then diffuse back into the stroma down this gradient through the channel enzyme ATP synthase.

As the protons pass through, the flow drives ATP synthase to catalyse the synthesis of ATP from ADP and inorganic phosphate (Pi). This mechanism is called chemiosmosis, and because it is driven by light it is photophosphorylation.

(Synoptic link: the same chemiosmotic mechanism produces most ATP in aerobic respiration in the mitochondria, and ATP acts as the universal energy currency of the cell.)

Fixation. Carbon dioxide is fixed by combining with ribulose bisphosphate (RuBP, 5C), catalysed by rubisco, to form two molecules of glycerate-3-phosphate (GP, 3C).

Reduction. Each GP is reduced to triose phosphate (GALP, 3C). This step uses reduced NADP (which provides hydrogen and is re-oxidised to NADP) and ATP (which provides energy and is hydrolysed to ADP and Pi) — both supplied by the light-dependent reactions.

Regeneration. Most of the triose phosphate is used to regenerate RuBP, using further ATP, so the cycle can continue to fix more carbon dioxide.

The remaining triose phosphate is used to make the organic products of photosynthesis — glucose, and from it starch, sucrose, cellulose, lipids and amino acids. In this way the ATP and reduced NADP made in the light reactions are 'spent' driving carbon fixation in the stroma.

Each turn of the Calvin cycle fixes only one molecule of carbon dioxide (1 carbon), but a molecule of glucose contains 6 carbons. Therefore six CO₂ molecules — and so six turns of the cycle — are needed to supply enough carbon for one glucose.

In each turn, one CO₂ combines with RuBP to make two GP (3C each), so six turns produce 12 GP, which are reduced to 12 triose phosphate (TP, 3C).

Of these 12 TP, two TP (2 × 3C = 6C) are combined to form one molecule of glucose (6C).

The remaining 10 TP are used to regenerate the six RuBP acceptor molecules (6 × 5C = 30 carbons = 10 × 3C), using ATP, so the cycle can continue.

This accounting (6 CO₂ in, 2 TP out as glucose, 10 TP recycled to RuBP) shows why six turns are required for a single hexose sugar.

When the carbon dioxide is removed, the concentration of GP falls and the concentration of RuBP rises.

GP is produced by the fixation of CO₂ onto RuBP (catalysed by rubisco). With no CO₂ available, no more GP can be formed by fixation. Meanwhile the light reactions continue (the light is still on), so ATP and reduced NADP keep being made and the existing GP continues to be reduced to triose phosphate. Because GP is still being used up but is no longer being made, its concentration falls.

At the same time, RuBP is still being regenerated from triose phosphate (using ATP), but it is no longer being consumed by reaction with CO₂ in the fixation step. Therefore RuBP is being made but not used, so its concentration rises / accumulates.

In summary: removing CO₂ blocks the fixation step. GP (downstream of fixation) decreases because it is used but not replaced; RuBP (the substrate of fixation) increases because it is regenerated but not consumed.

In non-cyclic photophosphorylation, electrons flow in one direction through both photosystem II and photosystem I. The electrons originate from the photolysis of water, so this pathway also splits water and releases oxygen. As electrons pass along the electron transport chain, H⁺ are pumped across the thylakoid membrane and ATP is made by chemiosmosis; at the end, electrons and H⁺ are used to reduce NADP. The products are therefore ATP, reduced NADP and oxygen.

In cyclic photophosphorylation, only photosystem I is used. The excited electrons from PSI are not passed to NADP but are instead returned to PSI via electron carriers, pumping H⁺ and generating ATP as they go. Because the electrons cycle back, there is no photolysis of water, no oxygen released and no reduced NADP formed — the only product is ATP.

Why cyclic is useful. The Calvin cycle uses more ATP than reduced NADP (ATP is needed in both the reduction and regeneration steps). Non-cyclic photophosphorylation alone may not make enough ATP relative to reduced NADP. Cyclic photophosphorylation lets the chloroplast make extra ATP without making more reduced NADP, topping up the ATP supply to keep the Calvin cycle running and to meet other ATP demands.

(a) Light intensity obeys the inverse square law: it is inversely proportional to the square of the distance from the lamp, $\text{light intensity} \propto \dfrac{1}{d^{2}}$. So as the lamp is moved further away, the light intensity falls rapidly — doubling the distance reduces the intensity to a quarter.

(b) Moving the lamp from 10 cm to 20 cm doubles the distance, so by the inverse square law the light intensity falls to $\left(\tfrac{1}{2}\right)^{2} = \tfrac{1}{4}$ of its original value. Provided light intensity is the limiting factor, the rate of photosynthesis would fall to about a quarter: $24 \div 4 = \textbf{6 bubbles per minute}$.

(c) Evaluation. Counting bubbles is not very reliable or valid because bubbles vary in size (so the same count may represent different volumes of oxygen), bubbles may form too fast to count accurately at high light intensity, and some oxygen dissolves in the water rather than forming bubbles. The method also assumes the gas is pure oxygen.

Improvement. Collect the gas in a capillary tube / gas syringe (photosynthometer) and measure the length or volume of oxygen produced per unit time instead of counting bubbles — this gives a more accurate, quantitative measure. (A heat-absorbing water screen between lamp and plant should also be used so that temperature is kept constant and only light intensity varies.)

(a) The Rf value is the distance moved by the pigment divided by the distance moved by the solvent front:

$R_f = \frac{\text{distance moved by pigment}}{\text{distance moved by solvent front}} = \frac{89}{95} = 0.9368\ldots \approx 0.94$

So the Rf value of carotene is 0.94 (no units).

(b) Carotene moved further up the paper (89 mm) than chlorophyll b (38 mm), giving carotene a higher Rf value ($89/95 = 0.94$ versus $38/95 = 0.40$). This is because carotene is more soluble in the (non-polar) solvent and is less strongly attracted to (adsorbed by) the chromatography paper, so it is carried further by the solvent front. Chlorophyll b is less soluble / more strongly retained by the paper, so it travels a shorter distance and has a lower Rf.