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7.3.2.2. Biosynthetic or dark phase

Biosynthetic or dark phase of photosynthesis

Although this phase that includes the reactions that reduce and assimilate CO2  is still called the “dark phase”, it is not very correct, since some enzymes responsible for these reactions, such as phosphatases, kinases and dehydrogenases, need to be stimulated by energy light.

The purpose of the biosynthetic phase is the synthesis of organic matter from inorganic matter, using the energy ( ATP ) and the reducing power ( NADPH ) obtained in the photochemical phase. It can be divided into:

  • Synthesis of carbon compounds: it is carried out in the stroma through the Calvin cycle, where CO2 is fixed, using ATP and NADPH obtained in the light phase, which can continue the cycle or follow other biosynthetic pathways. For every CO2 molecule that binds, three ATP and two NADPH are used up .   
  • Synthesis of nitrogenous organic compounds: from NADPH and ATP obtained in the light phase, the nitrate ions found in the soil are reduced to nitrite ions, and then to ammonia.
  • Synthesis of organic compounds with sulfur: from NADPH and ATP obtained in the light phase, the sulfate ion is reduced to sulfite ion and then to hydrogen sulfide.

By the reactions of the photochemical phase, the cells capture light energy and convert it into electrical energy (electron flow) and the electrical energy is transformed into chemical energy  stored in the bonds of NADPH and ATP.

In the biosynthetic phase , this energy is used to reduce carbon and synthesize carbohydrates . Photosynthetic cells take carbon from carbon dioxide, which in algae is dissolved in water, and in plants, through stomata .

Although these reactions occur regardless of the presence or not of light, they require NADPH and ATP, which can only be formed if there is light, so it is not entirely correct to refer to the biosynthetic phase as the dark phase.

The reduction of carbon occurs in the stroma of the chloroplast, where cyclical reactions called the Calvin cycle take place in honor of its discoverer, Melvin Calvin. 

Calvin cycle or pentose cycle: Three-carbon pathway

In the Calvin cycle, as in the Krebs cycle, the initial compound, after one turn, regenerates. In the Calvin cycle, the molecule that initiates the cycle is a five-carbon carbohydrate with two phosphate groups, ribulose-1,5-bisphosphate (RuBP).

The RuBP molecule (5 carbons) binds to a carbon dioxide molecule (1 carbon) and they separate rapidly forming two 3-phosphoglyceric acid (or PGA ) molecules (3 carbons), of which one contains the molecule of CO2 that has just been bonded.

Since 3-phosphoglyceric acid was one of the first molecules identified in this cycle, the Calvin cycle is also called the C3 or three-carbon pathway .

It is in the stroma of the chloroplast, with the participation of the enzyme RuBisCO, where the binding of RuBP with CO2 and its cleavage into two PGA molecules occurs.

The name of RuBisCO is the abbreviated form of the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase, which catalyzes two opposite processes:

  • The fixation of CO2 (carboxylase).
  • The photorespiration, which acts as oxygenase the same substrate.

The Rubisco is the most abundant protein of the Earth (15% of all proteins present in the chloroplast). It has to be very abundant because it can only bind three CO2 molecules per second.

The cycle begins when 3 CO2 molecules bind to ribulose-1,5-bisphosphate (RuBP) and separate into 6 PGAs, to later be reduced to glyceraldehyde-3-phosphate (GAP), using the NADPH and ATP formed in the electronic transport chain of the photochemical phase of photosynthesis .  

The transformation from PGA to GAP consists of two steps:

  • First, the six PGA molecules are phosphorylated to form six 1,3-bisphosphoglyceric acid (BPG) molecules using six ATP molecules.
  • The BPG is then reduced with the electrons it receives from NADPH, to form glyceraldehyde-3-phosphate (GAP).

Of the six molecules obtained from GAP, only one of them is used to synthesize carbohydrates in the cytosol, so it can be considered the product of light-independent reactions. The other five GAP molecules that have been formed will re-transform into three ribulose-1,5-bisphosphate (RuBP) molecules, which will rejoin other CO2 molecules in another round of the cycle. To produce RuBP again, the energy of three ATP molecules is needed. 

The glyceraldehyde 3-phosphate will be used to form glucose or other carbohydrates. In addition, they can also go to the cytoplasm and enter the Krebs cycle to provide energy, or stay in the chloroplast to synthesize carbohydrates, lipids, amino acids or nitrogenous bases . Remember that we have seen that glyceraldehyde phosphate was formed when the fructose bisphosphate molecule was transformed in the fourth step of glycolysis.  

Energy balance

The synthesis of carbohydrates entails a high energy expenditure. To fix 3 molecules of CO2 and produce 1 of GAP, 9 of ATP and 6 of NADPH are necessary. Or what is the same, to fix 6 molecules of CO in a molecule of 6 carbons like glucose, and regenerate ribulose-1,5-bisphosphate requires 12 molecules of NADPH and 18 molecules of ATP.

The overall reaction of the biosynthetic phase of photosynthesis is:

6 CO2  + 12 NADPH + 12 H+  + 18 ATP → 1 Hexose + 12 NADP+  + 18 ADP + 18 Pi

CO2 + H2O + light → C6H12O6

The reactions that take place in the Calvin cycle occur in all algae and in most plants, being the only way for their cells to transform the inorganic carbon in the air into organic molecules necessary for life.

At each turn of the Calvin cycle, a molecule of carbon dioxide enters, a molecule of RuBP is reduced and regenerated. It takes three rounds of the cycle to obtain a 3-carbon carbohydrate, glyceraldehyde phosphate. To obtain a 6-carbon carbohydrate, such as glucose, 6 molecules of carbon dioxide are required to enter the cycle.

The global equation is:

6RuBP + 6CO 2  + 18ATP + 12 NADPH + 12H +  + 12H 2 O → 6RuBP + glucose + 18P i  + 18ADP + 12 NADP +

Summary of the Calvin Cycle

At each turn of the cycle, a carbon dioxide molecule is introduced. In this scheme three turns are represented, those necessary to synthesize a molecule of glyceraldehyde phosphate (3 carbons). Three molecules of ribulose bisphosphate (RuBP), (3 x 5 carbons), combine with three of carbon dioxide (3 x 1 carbons), producing six molecules of phosphoglycerate (6 x 3 carbons). These molecules are reduced to six molecules of glyceraldehyde phosphate. Five of these three-carbon molecules combine and rearrange to form three five-carbon molecules of RuBP. The other molecule of glyceraldehyde phosphate is the product of the Calvin cycle.

At each turn of the Calvin cycle, a molecule of carbon dioxide enters, a molecule of RuBP is reduced and regenerated. It takes three rounds of the cycle to obtain a 3-carbon carbohydrate, glyceraldehyde phosphate. To obtain a 6-carbon carbohydrate, such as glucose, 6 molecules of carbon dioxide are required to enter the cycle.

The global equation is:

6RuBP + 6CO2 + 18ATP + 12NADPH + 12H+ + 12H2O → 6RuBP + glucose + 18Pi + 18ADP + 12 NADP+

Photorespiration

If the weather becomes warmer and the plants close their stomata to prevent water loss, the concentration of O2 increases  and that of CO2 within the leaf decreases. Photorespiration then occurs.

The enzyme RuBisCO, the ribulose-1,5-bisphosphate carboxylase oxygenase, intervened in fixing CO2, being carboxylase, but being oxygenase, also involved in photorespiration reversing operation, setting O2 and releasing CO2, oxidizing the ribulose-bisphosphate by adding O2 to it, which will cause CO2 and H2O, which is an energy expenditure for the plant.

The plant thus protects itself from photo-oxidation, although photorespiration harms it, since it reduces the photosynthetic capacity of the plant as CO2 and O2 compete for the active site of RuBisCO and energy is lost.

Four-carbon pathway

The plants of tropical climates have solved this problem because their leaves have two different types of cells, which allows them to have open stomata, but we will not see it this course.

Fundamental ideas about the biosynthetic phase of photosynthesis

Biosynthetic phase of photosynthesis

  • Organic matter is synthesized from inorganic matter, using the energy ( ATP ) and the reducing power ( NADPH ) obtained in the photochemical phase.
  • It takes place in the stroma of the chloroplast.
  • 6RuBP + 6CO + 18ATP + 12NADPH + 12H + 12H2O → 6RuBP + glucose + 18P + 18ADP + 12 NADP +
  • The  Rubisco   (enzyme ribulose-1,5-bisphosphate carboxylase oxygenase), which catalyzes two opposite processes:
    • The fixation of CO2 (carboxylase).
    • The photorespiration, which acts as oxygenase the same substrate.