Ribulose 1,5- bisphosphate carboxylase/oxygenase (RuBisCo) is a critical enzyme that catalyzes carbon dioxide assimilation during photosynthesis. However, it is an inefficient enzyme, having an extremely slow catalytic rate. A typical enzyme can process about a thousand molecules per second; however, RuBisCo fixes only around three-carbon dioxides per second. Photosynthetic cells compensate for this slow rate by synthesizing very high amounts of RuBisCo, making it the most abundant single enzyme on Earth.

In addition, RuBisCo has a poor substrate specificity due to which oxygen can easily attach to the carbon dioxide binding site of the enzyme. As a result, an abnormal molecule is produced along with the release of CO₂. This process is called photorespiration or, more accurately- oxidative photosynthetic carbon cycle.

Every photosynthetic organism experiences a basal level of photorespiration; however, under high intracellular oxygen levels, photorespiration exceeds photosynthesis. Most tropical plants have developed a mechanism to circumvent the wasteful photorespiration through a special process that increases the intracellular CO₂ levels. In such plants, the Calvin cycle's usual carbon fixation step is preceded by several steps that temporarily fix CO₂ by forming four-carbon intermediates such as oxaloacetate and malate. The plants that rely on this process are called C4 plants, and the assimilation process is termed the C4 pathway.

Another variation of CO₂ fixation is observed in succulent plants that grow in hot and arid environments. In these plants, stomata remain closed during the daytime to prevent loss of water from the plants. Because closure of stomata also prevents gas molecules from entering the leaf, CO₂ is absorbed during night-time when cool and moist air enables opening of stomata. CO₂ trapped overnight in the form of malate is released during the daytime by the NADP-linked malic enzymes. Because this method of CO₂ assimilation was first discovered in the plants of the Crassulaceae family, it is called crassulacean acid metabolism, or CAM pathway.