The Calvin Cycle

Every carbon atom in your body was, at some point, pulled out of the air as carbon dioxide by a plant, an alga or a cyanobacterium. The pathway that does this, turning an inorganic gas into sugar, is the Calvin cycle. It runs in the stroma of chloroplasts, powered by the ATP and NADPH made in the light-dependent reactions, and its central enzyme, RuBisCO, fixes on the order of a hundred billion tonnes of carbon every year.

How the pathway was discovered

In the late 1940s and 1950s, Melvin Calvin, Andrew Benson and James Bassham at Berkeley used the newly available radioactive isotope carbon-14:

1. They gave single-celled green algae a short burst of ¹⁴CO₂ while they photosynthesised.
2. After a few seconds, they dropped the algae into boiling alcohol, killing them instantly and stopping all reactions.
3. They separated the compounds in the extract by two-dimensional paper chromatography (see paper chromatography) and found which spots were radioactive by laying the paper on X-ray film.

After just a couple of seconds, most of the radioactivity was in one compound: 3-phosphoglycerate (3-PG). After longer times, it spread to sugar phosphates and other molecules. By varying the time and following the label, they traced the whole pathway. Calvin received the 1961 Nobel Prize in Chemistry. It’s a textbook example of using isotopes as tracers, the same idea behind carbon-14 dating.

The overall reaction

For every three molecules of CO₂ fixed, the cycle produces one molecule of glyceraldehyde-3-phosphate (G3P), a three-carbon sugar phosphate:

3CO₂ + 9ATP + 6NADPH + 6H⁺ → G3P + 9ADP + 8Pᵢ + 6NADP⁺ + 3H₂O

Making one six-carbon glucose requires two G3P, so 6CO₂, 18ATP and 12NADPH.

Phase 1: Carbon fixation

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, mercifully shortened to RuBisCO, attaches CO₂ to a five-carbon sugar, ribulose-1,5-bisphosphate (RuBP):

RuBP (5C) + CO₂ (1C) → unstable 6C intermediate → 2 × 3-phosphoglycerate (3C)

The six-carbon intermediate immediately splits into two molecules of 3-PG, which is why 3-PG appeared first in Calvin’s experiments. This step turns inorganic carbon into organic carbon, and it’s where CO₂ enters the living world.

For three turns of the cycle: 3 RuBP + 3 CO₂ → 6 3-PG.

Phase 2: Reduction

3-PG is a carboxylic acid, fairly oxidised. It’s reduced to a sugar (an aldehyde) in two steps:

1. Phosphorylation: ATP adds a phosphate to the carboxyl group, making 1,3-bisphosphoglycerate. (6 ATP used for 6 molecules.)
2. Reduction: NADPH reduces it to glyceraldehyde-3-phosphate (G3P), releasing phosphate. (6 NADPH used.)

These are essentially steps 6 and 7 of glycolysis run in reverse, using NADPH instead of NADH. This is the phase where the energy captured from light is stored as chemical energy in sugar.

After this phase: 6 G3P.

Phase 3: Regeneration of RuBP

Of the 6 G3P made, only 1 leaves the cycle as product. The other 5 (5 × 3 = 15 carbons) are rearranged through a series of reactions into 3 RuBP (3 × 5 = 15 carbons), using 3 more ATP. This regenerates the CO₂ acceptor so the cycle can continue.

The rearrangements shuffle carbon atoms between sugars of three, four, five, six and seven carbons, using enzymes such as transketolase and aldolase, before arriving back at five-carbon RuBP.

The budget

Phase	Per 3 CO₂	ATP used	NADPH used
Fixation	3 RuBP + 3 CO₂ → 6 3-PG	0	0
Reduction	6 3-PG → 6 G3P	6	6
Regeneration	5 G3P → 3 RuBP	3	0
Net	1 G3P produced	9	6

The 3:2 ratio of ATP to NADPH is why chloroplasts sometimes run cyclic electron flow to make extra ATP.

What happens to G3P

G3P leaves the cycle and is used to make:

• sucrose in the cytoplasm, for transport around the plant (see disaccharides);
• starch in the chloroplast, for storage (see starch vs glycogen vs cellulose);
• glucose and other sugars, fatty acids, and amino acids (with added nitrogen).

Regulation: switching on in the light

Although the Calvin cycle doesn’t use light directly, it’s switched on by light, so that the plant doesn’t waste ATP in the dark:

• In the light, protons are pumped into the thylakoids, so the stroma becomes more alkaline (about pH 8) and Mg²⁺ ions move into the stroma. RuBisCO works best under these conditions.
• Several Calvin cycle enzymes are activated by a system linked to the light reactions (through the protein thioredoxin), which reduces disulfide bridges in the enzymes.
• RuBisCO itself needs to be “activated” by a CO₂ molecule and Mg²⁺ binding to a lysine in its active site, helped by an enzyme called RuBisCO activase.

RuBisCO’s flaw: photorespiration

RuBisCO is a slow enzyme, handling only a few CO₂ molecules per second, so plants make enormous amounts of it: it can be up to half the soluble protein in a leaf, making it probably the most abundant protein on Earth.

It also has a flaw: it can react with O₂ instead of CO₂ (that’s the “oxygenase” in its name). When it does, RuBP is split into one 3-PG and one molecule of 2-phosphoglycolate, which is useless and must be recycled through a long pathway involving other organelles. This process, called photorespiration, uses energy and releases CO₂, undoing some of the plant’s work.

Photorespiration gets worse when:

• temperatures are high (RuBisCO’s preference for CO₂ over O₂ falls, and CO₂ becomes less soluble relative to O₂);
• stomata close in dry weather, so CO₂ inside the leaf drops and O₂ builds up.

Solutions: C4 and CAM plants

Some plants have evolved ways to concentrate CO₂ around RuBisCO:

• C4 plants (such as maize, sugar cane and sorghum) first fix CO₂ in outer leaf cells using a different enzyme (PEP carboxylase, which doesn’t react with O₂), forming a four-carbon acid. This is shuttled to inner cells, where CO₂ is released at high concentration right next to RuBisCO. C4 plants do especially well in hot, sunny climates.
• CAM plants (such as cacti and pineapples) open their stomata only at night, fixing CO₂ into acids that are stored overnight. By day, with stomata closed to save water, they release the CO₂ internally for the Calvin cycle. This is a separation in time rather than space.

Scientists are working to engineer more efficient RuBisCO or C4-like systems into crops such as rice to increase yields.

Key takeaways

• The Calvin cycle runs in the stroma and uses ATP and NADPH from the light reactions to turn CO₂ into sugar.
• Fixation: RuBisCO adds CO₂ to RuBP (5C), giving two 3-phosphoglycerate (3C).
• Reduction: ATP and NADPH convert 3-PG into G3P; regeneration rebuilds RuBP using more ATP.
• Per 3 CO₂: 9 ATP + 6 NADPH → 1 G3P; per glucose: 18 ATP + 12 NADPH.
• RuBisCO also reacts with O₂ (photorespiration); C4 and CAM plants concentrate CO₂ to avoid it. For the overview, see photosynthesis: the chemistry.