Photosynthesis: Pathway of Carbon Fixation

Photosynthesis is the synthesis of organic molecules using the energy of light. For the sugar glucose (one of the most abundant products of photosynthesis) the equation is:
6CO₂ + 12H₂O -> C₆H₁₂O₆ + 6H₂O + 6O₂
A description of the experiments that led to this equation are described in Discovering the Secrets of Photosynthesis.
Light provides the energy to:

• transfer electrons from water to nicotinamide adenine dinucleotide phosphate (NADP⁺) forming NADPH;
• generate ATP.

The details of these processes are described in Photosynthesis: The Role of Light.
ATP and NADPH provide the energy and electrons to reduce carbon dioxide (CO₂) to organic molecules.

The Steps

• CO₂ combines with the phosphorylated 5-carbon sugar ribulose bisphosphate.
• This reaction is catalyzed by the enzyme ribulose bisphosphate carboxylase oxygenase (RUBISCO)(an enzyme which can fairly claim to be the most abundant protein on earth).
• The resulting 6-carbon compound breaks down into two molecules of 3-phosphoglyceric acid (PGA).
• The PGA molecules are further phosphorylated (by ATP) and are reduced (by NADPH) to form phosphoglyceraldehyde (PGAL).
• Phosphoglyceraldehyde serves as the starting material for the synthesis of glucose and fructose.
• Glucose and fructose make the disaccharide sucrose, which travels in solution to other parts of the plant (e.g., fruit, roots).
• Glucose is also the monomer used in the synthesis of the polysaccharides starch and cellulose.

The graphic shows the steps in the fixation of carbon dioxide during photosynthesis. All of these reactions occur in the stroma of the chloroplast.

Link to chloroplasts

These steps were worked out by Melvin Calvin and his colleagues at the University of California and, for this reason, are named the Calvin cycle.

Their Experiment

Apparatus used to follow the fate of 14CO2 in the dark reactions. The algal suspension is placed in the "lollypop", supplied with 14CO2, and illuminated. The dark reactions are halted by draining the contents of the lollypop into a flask of hot alcohol. (Courtesy of Dr. James A. Bassham.)

The experimental apparatus is shown at the right. After various intervals of illumination, a suspension of unicellular algae is inactivated and the contents of the cells extracted. The compounds in a drop of the extract are then separated by paper chromatography.
The identity of each substance may be determined simply by comparing its position with the positions occupied by known substances under the same conditions. Or, a fragment containing the spot can be cut from the sheet and chemically analyzed.
To determine which, if any, of the substances separated on the chromatogram are radioactive, a sheet of X-ray film is placed next to the chromatogram. If dark spots appear on the film (because of radiation emitted by the ¹⁴C atoms), their position can be correlated with the positions of the chemicals in the chromatogram. Using this technique of autoradiography, Calvin found that ¹⁴C turned up in glucose molecules within 30 seconds after the start of photosynthesis. When he permitted photosynthesis to proceed for only 5 seconds, however, the radioactivity was concentrated in several other, smaller, molecules.

The dark spots show the radioactive compounds produced after 10 secs (left) and 2 minutes (right) of photosynthesis by the green alga Scenedesmus. The alga was supplied with carbon dioxide labeled with 14C, a radioactive isotope of carbon. At 10 seconds, most of the radioactivity is found in 3-phosphoglyceric acid ("P-Glyceric"). At 2 minutes, phosphorylated 6-carbon sugars (glucose and fructose) have been synthesized as well as a number of amino acids. The small rectangle and circle (lower right-hand corners) mark the spots where the cell extract was applied. (Courtesy of Dr. James A. Bassham.)

Link to discussion of the free energy changes in photosynthesis.

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