Perhaps no chemical process has a greater impact on life than the conversion of atmospheric carbon dioxide and water into carbohydrates by photosynthetic plants. Photosynthesis can be divided into two major reaction pathways. The first pathway, driven by light energy from the sun, uses electron transport and photophosphorylation to produce ATP and NADPH. These energy-rich compounds are then utilized in the second pathway to convert carbon dioxide into carbohydrate molecules—a process called carbon fixation. This latter pathway, called the Calvin cycle, was first elucidated by Melvin Calvin and colleagues at UC Berkeley.
Photosynthetic reactions take place in the chloroplast and can be divided into two pathways. Within the thylakoid membrane, the energy-transduction pathway converts light energy into ATP and NADPH. The second pathway takes place in the stroma, and uses energy produced by the first pathway to incorporate carbon dioxide into carbohydrates in a process called carbon fixation.
The sequence of reactions by which carbon dioxide is converted into carbohydrates was uncovered in the 1940s and 1950s by Melvin Calvin, Andrew Benson, and colleagues at UC Berkeley. How did these scientists determine the pathway of carbon dioxide fixation in photosynthesis?
Like other researchers, Calvin and Benson were interested in the basic photosynthetic process of using light energy to convert carbon dioxide and water into carbohydrates and oxygen. However, there was no obvious way to follow the path taken by the carbon contained in carbon dioxide as it became incorporated into other molecules.
In the mid-1940s, however, nuclear reactors were being built, and scientists could use these reactors to create radioactive isotopes of common atoms like carbon. In nature, carbon exists as the 12C isotope, but a radioactive isotope of carbon, 14C, could now be produced that behaved almost identically to 12C. A useful tool was thus available for tracing the path of carbon dioxide in photosynthesis.
Calvin and Benson first constructed an apparatus consisting of a light source and a flask that could be filled with photosynthetic organisms. For this purpose, the researchers chose the unicellular green algae, Chorella.
To label metabolites, the researchers injected 14C-labeled CO2 into the flask, and then quickly killed the algae and extracted their metabolites in boiling ethanol.
Using a technique called two-dimensional paper chromatography, the researchers were able to separate the components they sought. In this technique, the plant extract is first spotted in one corner of a piece of filter paper. The paper is then suspended in a chamber containing a solvent in such a way that the solvent just touches the edge of the paper.
Capillary action will pull the solvent across the paper. As it passes over the extract, it will pick up various components. Based on their molecular properties, the components will travel at different speeds as they are pulled along the piece of paper by the solvent. In order to improve the separation even more, a second run was performed in a perpendicular direction.
By placing an x-ray film on top of the paper, the locations of the radioactivity-labeled compounds could be determined. When the Chorella was exposed to 14C-labeled CO2 for 30 seconds, a variety of compounds were found to be labeled, including organic compounds, amino acids, and sugars. All of these compounds had incorporated carbon from the 30-second exposure to 14C-labeled CO2.
But the authors wanted to determine the first compounds to become labeled, so they continued to reduce the reaction time until only a single compound dominated the chromatogram. This compound, which became labeled after just a few seconds of exposure, was identified as a three-carbon sugar phosphate called 3-phosphoglycerate (3PG).
The researchers eventually identified the acceptor molecule for carbon dioxide as the five-carbon compound ribulose bisphosphate (RuBP). Catalyzed by the abundant enzyme ribulose bisphosphate carboxylase/oxygenase (or rubisco), the carbon dioxide becomes incorporated into the RuBP molecule, which is then converted into two molecules of 3-phosphoglycerate.
Using a combination of chromatographic and chemical techniques, Calvin and his colleagues eventually identified all of the intermediates within the cycle, and Melvin Calvin was awarded the Nobel prize for his work in 1961.
Prior to 1940, investigating the chemical reactions involved in carbon fixation was hindered by the fact that it was difficult to distinguish the carbon atoms present in atmospheric CO2 from those contained in the substrates and products of the photosynthetic reactions. The subsequent development of radioactive tracers provided a tool for scientists to examine these pathways. By labeling carbon dioxide molecules using a heavy isotope of carbon, Calvin and colleagues were able to trace the integration of atmospheric carbon in the form of CO2 into a variety of compounds by photosynthetic organisms. By combining tracer labeling with paper chromatography, these researchers were able to establish ribulose 1,5-bisphosphate (RuBP) as the carbon dioxide acceptor molecule, as well as to elucidate the entire pathway of carbon fixation, sugar production, and regeneration of RuBP.
In recognition of the importance of these discoveries, Melvin Calvin was awarded the Nobel Prize in chemistry in 1961.