C₄ Carbon Fixation - C₄ Pathway

C₄ Pathway

The first experiments indicating that some plants do not use the established C3 carbon fixation but produce malate and aspartate in the first step were done in the 1950s and early 1960s by Hugo P. Kortschak and Yuri Karpilov The C₄ pathway was finally discovered by Marshall Davidson Hatch and C. R. Slack, in Australia, in 1966, so it is sometimes called the Hatch-Slack pathway.

In C₃ plants, the first step in the light-independent reactions of photosynthesis involves the fixation of CO₂ by the enzyme RuBisCO into 3-phosphoglycerate. However, due to the dual carboxylase / oxygenase activity of RuBisCo, an amount of the substrate is oxidized rather than carboxylated, resulting in loss of substrate and consumption of energy, in what is known as photorespiration. In order to bypass the photorespiration pathway, C₄ plants have developed a mechanism to efficiently deliver CO₂ to the RuBisCO enzyme. They utilize their specific leaf anatomy where chloroplasts exist not only in the mesophyll cells in the outer part of their leaves but in the bundle sheath cells as well. Instead of direct fixation to RuBisCO in the Calvin cycle, CO₂ is incorporated into a 4-carbon organic acid, which has the ability to regenerate CO₂ in the chloroplasts of the bundle sheath cells. Bundle sheath cells can then utilize this CO₂ to generate carbohydrates by the conventional C₃ pathway.

The first step in the pathway is the conversion of pyruvate to phosphoenolpyruvate (PEP), by the enzyme pyruvate orthophosphate dikinase. This reaction requires inorganic phosphate and ATP plus pyruvate, producing phosphoenolpyruvate, AMP, and inorganic pyrophosphate (PPi). The next step is the fixation of CO₂ into oxaloacetate by the enzyme PEP carboxylase. Both of these steps occur in the mesophyll cells:

pyruvate + Pi + ATP → PEP + AMP + PPi

PEP + CO₂ → oxaloacetate

PEP carboxylase has a lower Km for CO₂ — and, hence, higher affinity — than RuBisCO. Furthermore, O₂ is a very poor substrate for this enzyme. Thus, at relatively low concentrations of CO₂, most CO₂ will be fixed by this pathway.

The product is usually converted to malate, a simple organic compound, which is transported to the bundle-sheath cells surrounding a nearby vein. Here, it is decarboxylated to produce CO₂ and pyruvate. The CO₂ now enters the Calvin cycle and the pyruvate is transported back to the mesophyll cell.

Since every CO₂ molecule has to be fixed twice, first by 4-carbon organic acid and second by RuBisCO, the C₄ pathway uses more energy than the C₃ pathway. The C₃ pathway requires 18 molecules of ATP for the synthesis of one molecule of glucose, whereas the C₄ pathway requires 30 molecules of ATP. This energy debt is more than paid for by avoiding losing more than half of photosynthetic carbon in photorespiration as occurs in some tropical plants, making it an adaptive mechanism for minimizing the loss.

There are several variants of this pathway:

1. The 4-carbon acid transported from mesophyll cells may be malate, as above, or aspartate
2. The 3-carbon acid transported back from bundle-sheath cells may be pyruvate, as above, or alanine
3. The enzyme that catalyses decarboxylation in bundle-sheath cells differs. In maize and sugarcane, the enzyme is NADP-malic enzyme; in millet, it is NAD-malic enzyme; and, in Panicum maximum, it is PEP carboxykinase.