This part of photosynthesis occurs in the stroma of the chloroplasts called carbon dioxide fixation.

The fixation of the CO₂ is carried out by a giant enzyme ribulose biphosphate carboxylase/oxidase (RUBISCO) which is the most abundant enzyme on earth. This enzyme is very sluggish it works much slower than most other enzymes. (i.e. ~ 3 molecules of substrate per sec. compared with ~1000/sec for others). Therefore, there are many copies of this enzyme in the stroma ~ 50% of chloroplast protein.

The first fixation reaction of the cycle uses a five carbon sugar ribulose 1-5 biphosphate and adds to it a CO₂ molecule to form 2 (3 carbon) molecules of 3 - phosphoglycerate. These are rearranged through a series of energy requiring reactions, using up ATP and NADPH to generate 2 molecules of glyceraldehyde 3 - phosphate. (If this were done six (6) times we now would have 12 molecules of glyceraldehyde - 3 - phosphate (G3P). Two (2) of the G3Ps are removed to make one glucose while the rest 10 G3Ps go back into the cycle to regenerate six (6) of the five (5) carbon sugars ribulose 1-5 biphosphate.

Plants also make other sorts of molecules from the products of the Calvin cycle. For example G3P is used by most seed plants to fashion a number of lipids and amino acids as well as Nitrogen bases (DNA and RNA).

What factors effect how much glucose can be "fixed"?

Rate of photosynthesis can be limited by physical factors such as temperature. The lower the temperature the slower that rate. (Within the functional range of the photosynthetic enzymes).

Light availability can be a major factor in determining the rate of photosynthesis.

As the amount of light decrease the rate of photosynthesis declines. Eventually there is sufficient light intensity and photosynthesis becomes equal to respiration. This is referred to as the compensation point. (Respiration = photosynthesis).

E.g. Lakes - Light absorption by water. (Alternation)

In C-3 Plants in hot dry climates and in sunny conditions, the plants will close their stomata during the day to reduce the amount of water they lose through evaporation. This can cause the levels of CO₂ within the cells to drop RUBISCO will then add O₂ to the Calvin cycle rather then fix CO₂. This is known as light induced respiration (consumption of oxygen) or photorespiration.

Rate of photorespiration is stimulated by four factors:

1) high light levels,

2) high O₂ levels,

3) low CO₂ levels and

4) high temperatures

During normal CO₂ fixation in the Calvin cycle CO₂ is added to ribulose-biphosphate to form 2 molecules of 3 - phosphoglycerate (and on throught the cycle). The enzyme involved here was ribulose biphosphate carboxilase (ribisco).

However: In photorespiration rubisco can also take O₂ and attach it to ribulose-biphosphate instead of CO₂. Therefore rubisco can also function as an oxydase.

E.g.:

CO₂             rubisco

RuBP -------> 2(3PGA) -------> Calvin cycle

Or photorespiration

RuBP -------> 3 phosphoglyerate + P-glycolate

(5c)                         (3c)                             (2c)

What if rubisco evolved before O₂ became plentiful in the primitive earth atmosphere then due to its shape as O₂ built up in the atmosphere rubisco could not tell the difference between CO₂ and O₂? Some plants have developed additional metabolic pathways (C₄ and CAM) to reduce the fixation of O₂.

In certain climates sunlight is very abundant, and seldom if ever becomes limiting to photosynthesis. However, such climates as found in dry, hot regions can produce another limiting factor "CO₂".

One can think of it in terms of availability of H₂O and the loss of H₂O.

When the plant is photosynthesising in bright sun, the CO₂ must enter the leaves through the stomata (small holes in the underside of the leaf). But when these holes are "open" H₂O can also escape therefore, the plant dehydrates. If you close the stomata CO₂ becomes limiting.

In these C₄ plants, CO₂ is bound into phosphenol pyruvate (pep), (recall glycolysis) in cells in the leaf known as "mesophyll cells". As CO₂-pep and The CO₂ is released into the bundle sheath cells, which surround the vascular bundle. In these bundle sheath cells the CO₂ enters the Calvin cycle as usual.

In effect the mesophyll cells of a C₄ plant pump CO₂ into the bundle sheath cells, keeping the CO₂ concentration in the bundle sheath cells high enough for RUBISCO to fix CO₂ rather than Oxygen. In this way C4 plants can minimise photorespiration and maximise sugar production.

This system allows for spatial separation between the CO₂ absorbing C4 system and the Calvin cycle.

The ultimate prevention of CO₂ loss is found in desert plants like cactus. CAM In these plants the stomata are open at night. The plant fixes CO₂ into C₄ carbon compounds during the night, then transfers the carbon to the Calvin cycle during the daylight hours while the stomata are completely closed therefore reducing H₂O loss. This is all done in the same cell.