CO₂ Fixation

The anabolic or biosynthetic reactions are frequently termed 'Dark Reactions'. This is to indicate that they don't utilize light directly for energy, but rather the ATP and NADPH that are products of the photochemical reactions. However, these reactions don't really function in the dark because some of the key enzymes are regulated by light so that these reactions are not running at night and wasting cellular energy. It might be better to think of this phase of the biochemical process as the 'Biosynthetic Reactions'.

CO₂ is fixed [local] into organic matter in reactions using ATP and high energy e^- in the form of NADPH. This pathway is usually called the Calvin Cycle or the Calvin-Benson Cycle. Sometimes, this pathway is also called the Reductive Pentose Phosphate cycle.

The first step is catalyzed by Ribulose-1,5-bisPhosphate Carboxylase/Oxygenase (RuBisCO or rubisco). This enzyme is the most abundant protein on earth. Rubisco comprises ~50% dry weight of leaves. It has a complex quaternary structure, being composed of 8 large subunits (~55kD) and 8 small subunits (~15 kD). The active site is in the large subunits; the exact role of the small subunits isn't yet fully understood.

The reaction:

Ru-1,5-BP + CO₂ --> 2 molecules of 3-phosphoglycerate (also a C-3 glycolytic intermediate). Even though the RuBisCO reaction is converting a gas (CO₂) to a solid, the overall standard free energy is -35 kJ/mol; part of this is because there are two molecules as product. 3-phosphoglycerate is processed to produce carbohydrate and regenerate RuBP. Notice the roles of aldolase and transketolase in moving carbon atoms around to make sugars of different sizes. These two enzymes add much versatility to metabolism.

It is important that the Calvin cycle be regulated so that it does not operate in the dark. If it ran at night, it would rapidly deplete the cell of available energy and cause a lethal futile cycle. Regulation involves sensitivity to fluxes that increase stromal Mg²⁺ in the light and decrease it in the dark. Light also causes reduction of thioredoxin, a redox-sensing protein in the stroma of the chloroplast. When reduced in the light, thioredoxin activates several enzymes of the Calvin cycle [Fru-BPase, Sed-BPase, Ru-5-P kinase].

Rubisco can also catalyze addition of O₂ [local] to RuBP to make 3-phosphoglyceric acid + 2-phosphoglycolate (2 carbons). Phosphoglycolate is further metabolized in the peroxisome and the mitochondrion. During this process, a CO₂ and NH₃ are used up in the pathway, but phosphoglycerate is regenerated in the chloroplast to feed back into the Calvin cycle.

This process is photorespiration. It is wasteful. Perhaps 40% of the energy derived from photosynthesis is lost in photorespiration. We don't know if it serves a purpose.

Why hasn't Rubisco evolved a greater efficiency for CO₂ and less affinity for O₂? These are very small molecules, without much structural basis for discrimination. The active site would have a hard time developing an ability to discriminate without greatly slowing the reaction.

For some species, photorespiration is not a problem. In the past few thousand years they have evolved the capacity to fix CO₂ into C-4 acids [local] (e.g. OAA) first. The CO₂ is then released in specialized cells containing Rubisco. This process acts as a CO₂ pump to raise the CO₂ in the vicinity of the Rubisco and help it compete with O₂ in the active site.

 Another evolved variation is to use C4-type metabolism to fix CO₂ into organic acids at night. This is termed Crassulacean Acid Metabolism [local] and is typically found in desert plants. The organic acids are stored in the vacuole until morning, when photosynthetic energy production is used to drive the decarboxylation of these organic acids and refixation of the CO₂ by rubisco. The advantage for these plants is that leaf stomates can be kept closed during the day when water loss by evaporation and transpiration is the greatest. These plants are usually better adapted for growth under arid conditions.