You should have a working knowledge of the following terms:
- Calvin cycle
- carbon cycle
- glyceraldehyde 3-phosphate (G3P)
- ribulose bisphosphate (RuBP)
- stoma (pl. stomata)
Introduction and Goals
The last tutorial explored the light reactions of photosynthesis. These reactions begin with the absorption of light energy by pigments, and end with the production of stored chemical energy in the form of NADPH and ATP. Now we will follow the NADPH and ATP molecules as they enter the Calvin cycle. Their stored energy will be used to make sugar from carbon dioxide. These anabolic reactions are endergonic (have a positive delta G), and therefore require energy (from ATP and NADPH). The basic relationship between the Calvin cycle and the light reactions is summarized in this figure.
These reactions are sometimes called the "dark reactions" because they can occur in the dark (as long as ATP and NADPH are available). All of the processes of photosynthesis (light and dark reactions) occur within the chloroplast. By the end of this tutorial you should have a basic working understanding of:
- The energetics of photosynthesis
- The basic workings of the Calvin cycle (including the fixation, reduction, and regeneration phases)
Before we begin our study of the Calvin cycle, let's look at the energetics (total energy relations and transformations of a system) of sugar synthesis.
We have already studied the catabolism (breakdown) of glucose during cellular respiration. You know that the process is exergonic and releases about 686 kcal of energy. Thus, the delta G for the overall reaction is -686 kcal/mole.
glucose + O2 --> CO2 + H2O + ATP
If 686 kcal of energy per mole are released in the process of respiration, then it follows that 686 kcal of energy (minimum) are required to produce one mole of glucose. (Remember the first law of thermodynamics?) The Calvin cycle is the process by which glucose is made, and it requires all of that energy. Where does the energy come from? The light reactions of photosynthesis produce ATP, which provides the Calvin cycle with the necessary energy. In addition, the NADPH produced by the light reactions provides the reducing power to put glucose together.
sunlight + CO2 + H2O --> O2 + glucose
Overview of the Calvin Cycle
The light reactions of photosynthesis produce ATP and NADPH, which are then used in glucose synthesis during the Calvin cycle. As you should know from studying the Krebs cycle, metabolic cycles involve inputs and outputs, but some molecules are recycled to go full circle.
In the case of the Calvin Cycle, the input molecules are carbon dioxide, ATP, and NADPH. The output molecules are sugar, ADP, NADP+, and inorganic phosphate (Pi). The recycled molecule is ribulose bisphosphate (RuBP). Look at this figure and take a moment to locate these molecules.
Scientific knowledge is gained through observations and controlled experiments, but how does one study a process that can't be seen directly?
About 50 years ago, Melvin Calvin tried to do just that. He was working in a laboratory at the University of California, Berkeley during WWII. The study of radioactive elements had become an important new field in chemistry during the war. Among these newly discovered radioactive elements was carbon14. On the day in 1945 that the Japanese surrendered, a friend and colleague told Calvin, "Now is the time to do something useful with radioactive carbon." Calvin turned his focus to the study of photosynthesis, and 16 years later he won a Nobel Prize in chemistry and a metabolic process was named after him.
Calvin knew that photosynthesis could only occur in living organisms. Thus, the study of the chemical process was a difficult one. He devised a method whereby he could raise algae (Chlorella pyrenoidosa) in a lollipop-shaped disk (see photo). He set up a stream of air that could be controlled. He could inject radioactive carbon as carbon dioxide into the air stream for a set period of time. Then he would kill the algae with boiling methanol to stop the process of photosynthesis. He ran the experiment multiple times, each time killing the algae at different lengths of time after injecting carbon14.
Figure 3(Click image to enlarge)
Calvin analyzed the dead algae to determine which molecules had incorporated the carbon14. The technique he used is called chromatography. By comparing the molecules that contained carbon14 after each time period, he found a sequence of compounds that revealed the path of carbon dioxide as it was turned into glucose. In the years that followed, other researchers have discovered the enzymes and other compounds that also function in the Calvin cycle.
Carbon Dioxide Fixation Yields Two, 3-Carbon Compounds
Calvin first saw a three-carbon compound that was radioactively labeled. This led him to conclude that there was a two-carbon compound that was binding to the carbon dioxide, fixing it into an organic compound. However, when he stopped the process almost immediately after injecting carbon dioxide, he found a six-carbon compound. It turns out that carbon dioxide is initially fixed (i.e., taken out of the gas phase) by joining to a five-carbon compound. The resulting six-carbon compound is so unstable that it very quickly breaks into two, three-carbon compounds.
Figure 2(Click image to enlarge)
The top part of this diagram shows the fixation process. The five-carbon compound is ribulose bisphosphate (RuBP). An enzyme, rubisco (the most abundant protein on Earth), helps join one molecule of incoming carbon dioxide with one molecule of RuBP. The six-carbon compound that results immediately splits into two molecules, each with three carbons.
The carbon dioxide needed for this part of the Calvin cycle can get to the chloroplast in several different ways. In algae it simply diffuses through membranes from the surrounding water. In plants the carbon dioxide comes in through pores (stomata) in the leaves and uses one of several mechanisms to get to the chloroplasts. These same pores are where oxygen, produced in the light reactions, escapes into the atmosphere.
Reduction of the Two, Three-Carbon Compounds
In the first phase of the Calvin cycle, carbon dioxide is fixed into a 6-carbon molecule, which splits into two, 3-carbon molecules. In the second phase (shown in this figure), the 3-carbon molecules are reduced to glyceraldehyde 3-phosphate (G3P), another 3-carbon molecule. Remember, reduction means that electrons are added to the molecule. NADPH, produced during the light reactions, provides the high-energy electrons for this process. Also in this reduction phase, some ATP is used. Thus, the Calvin cycle is energetically tied to the light reactions of photosynthesis.
At the end of the reduction phase, some of the G3P leaves the cycle to become sugar, however, most of it gets regenerated into RuBP.
Regeneration of G3P to RuBP
As shown in these two figures, the Krebs cycle and the Calvin cycle have some general similarities. Remember, the Krebs cycle regenerates oxaloacetate at the end of one cycle to begin the next. In much the same way, the Calvin cycle regenerates RuBP to begin the next cycle. For every three carbon dioxide molecules that are fixed, three molecules of RuBP were needed. Thus, at the end of the cycle there must be three molecules of RuBP or the cycle would get out of balance.
The three molecules of RuBP that began the cycle had a total of three carbons multiplied by five molecules, or 15 atoms of carbon. Three molecules of carbon were then fixed for a surplus of three carbons in the cycle. Those three carbons are expelled from the cycle as one molecule of G3P. The remaining 15 carbons are still in the form of G3P. Therefore, they must be converted back to RuBP to start the process again. More ATP, as well as many steps involving enzymes, are necessary to do this regeneration.
Although we commonly think about the Calvin cycle occurring in this simple form, it is important to keep in mind the complexity of the cell. Our simplification of the Calvin cycle shows that a given chloroplast only has one enzyme for each step in the cycle. You need to remember that each chloroplast has a multitude of each type of enzyme. Thus, hundreds of molecules are going through this cycle at once. Before proceeding to the next page, click on the image of the Calvin cycle and note where NADPH is first used. The next question will ask what the consequences would be if NADPH levels were to suddenly drop. (In the absence of the reducing power of NADPH, what intermediate of the Calvin Cycle would begin to accumulate?)
Carbon dioxide is constantly being fixed into sugars (and other macromolecules), which, in turn, are oxidized back into CO2. This relationship, on a global scale, is termed the carbon cycle. However, humans are burning fossil fuels at a faster rate than plants can fix them back into sugars and other carbon molecules. Therefore, the global carbon cycle is out of balance. Or is it? Some scientists think that the current rise in CO2 levels is part of a natural cycle (rising and falling CO2 levels) that has been going on for millions of years. Thus, there are cycles within cycles, each interacting with and affecting the others. For more information on global warming, visit these sites:
We have finished our discussion of photosynthesis by showing the major anabolic pathway that results in the reduction of carbon dioxide to various sugars (including glucose). The formation of sugars is energetically unfavorable, as can be predicted from the positive ?G for the formation of glucose from carbon dioxide and water. The energy and reducing power for this process comes from the light-dependent reactions, which were presented in the last tutorial.
Basically, the Calvin cycle is a collection of enzymes that work to introduce carbon dioxide into the anabolic pathway involved in sugar production. Keep in mind, there are some variations on how carbon is initially fixed (depending on the species of photosynthetic organism), but they all have the enzymes that are involved in the Calvin cycle.
Carbon dioxide enters the Calvin cycle by being added to the 5-carbon compound called ribulose bisphosphate (RuBP). This reaction, involving the enzyme rubisco (the most abundant protein on Earth), results in a 6-carbon compound that is very unstable and is rapidly split into two, 3-carbon molecules. The resulting 3-carbon compound is activated by phosphorylation (via ATP obtained from the light-driven reactions of photosynthesis), then reduced by NADPH (again, obtained from the light-driven reactions of photosynthesis). After reduction, a 3-carbon molecule, glyceraldehyde 3-phosphate (G3P), exists. G3P has two fates. Some molecules go on to form more molecules of RuBP (which requires additional ATP; again derived from the light-driven photosynthetic reactions). Some molecules exit the Calvin cycle to form sugars (including glucose).
If you are curious about how sugars are formed from G3P, you might want to review the glycolytic pathway and find G3P. If you go backwards, you will see the pathway that is involved in the production of glucose. Other sugars can be made using other metabolic pathways.