Light Independent Stage Handout PDF

**[Light Independent Stage]** What can you remember from the light dependent stage? Th light independent stage of photosynthesis uses the reducing power (reduced NADP) and ATP produced by the light-dependent stage to build **carbohydrates**. This stage consists of a series of reactions known as...

**[Light Independent Stage]** What can you remember from the light dependent stage? Th light independent stage of photosynthesis uses the reducing power (reduced NADP) and ATP produced by the light-dependent stage to build **carbohydrates**. This stage consists of a series of reactions known as the **Calvin cycle** and takes place in the **stroma** of the chloroplast. It is comprised of a series of small steps resulting in the reduction of carbon dioxide from the air to bring about the synthesis of carbohydrates. Each stage of the cycle is controlled by enzymes. **[The Calvin Cycle]** In the first stage, carbon dioxide from the air combines with the 5-carbon compound ribulose bisphosphate (RuBP) in the chloroplasts to form a 6C compound. The carbon dioxide is said to be **fixed**, so this is **carbon fixation**. The enzyme **ribulose bisphosphate carboxylase/oxygenase** (usually known as RUBISCO) is necessary for this vital step. Research has shown that RUBISCO is the rate-limiting enzyme in the process of photosynthesis. 1. **[Formation of glycerate 3-phosphate]** Carbon dioxide enters the leaf through the stomata and diffuses into the stroma of the chloroplast. Here it's combined with ribulose bisphosphate (RuBP), a 5-carbon compound. This gives an unstable 6-carbon compound, which quickly breaks down into two molecules of a 3-carbon compound called glycerate 3-phosphate (GP). Ribulose bisphosphate carboxylase/oxygenase (RUBISCO) catalyses the reaction between carbon dioxide and RuBP. Research has shown this to be a rate limiting enzyme in photosynthesis. 2. **[Formation of glyceraldehyde 3-phosphate]** The 3-carbon compound GP is reduced (hydrogen is added) to form glyceraldehyde 3-phosphate. (GALP). The H^+^ ions come from reduced NADP and the energy comes from ATP, both produced in the light dependent stage. Glyceraldehyde 3-phosphate can then be converted into many useful organic compounds e.g. glucose. 3. **[Regeneration of Ribulose Bisphosphate]** **Much of the 3-carbon GALP produced in the cycle passes through a** **series of steps to replace the RuBP needed in the first step of the** **cycle.** **However, some of it is synthesised into the 6-carbon sugar glucose or passed** **directly into the glycolysis pathway where it may be used for the synthesis of other** **molecules needed by the plant.**  **[Products]** Diagram Description automatically generated GALP is the primary end-product of photosynthesis and it is the key molecule for the synthesis of everything else needed in the plant. - Some of the GALP is used directly in glycolysis and so fed on into the Krebs cycle. - Some of the GALP is used to produce glucose in a process called **gluconeogenesis**. - This glucose may then be converted into disaccharides such as sucrose for transport round the plant and into polysaccharides such as starch for energy storage and cellulose for structural support. - GALP that feeds into **Glycolysis** and Krebs cycle pathways is used to provide energy in the form of ATP for the functions of the cell. - Some of the GALP taken into the glycolysis pathway is converted to **acetyl coenzyme A**, which can be used to synthesise fatty acids needed for production of phospholipids for membranes, lipids. - Compounds from these pathways are also used as the building blocks of amino acids, combining the molecules with nitrates from the soil. - If GALP continues round the Calvin cycle, it can also be used for the production of nucleic acids with the addition of phosphates from the soil. **[RUBISCO and photorespiration]** RUBISCO makes up about 30% of the total protein of a leaf, so it is probably the most common protein on Earth. It is also possibly the most important enzyme because of its role in fixing carbon dioxide during photosynthesis. But RUBISCO is very inefficient. The active site cannot distinguish between the carbon- oxygen double bonds of CO~2~ and the oxygen-oxygen double bonds of O­~2~. As a result there is competitive inhibition between the two. +-----------------------------------+-----------------------------------+ | **RUBISCO as a carboxylase** | **RUBISCO as an oxygenase** | | | | | In high levels of CO­~2~ / | In low levels of CO~2~/relatively | | relatively low O~2~, RUBISCO | high O~2,~ RUBISCO binds to the | | binds to the carbon dioxide and | oxygen and combines it with RuBP | | combines it with RuBP, giving two | to form one molecule of GP and | | molecules of 3C GP which feeds | one molecule of | | into the Calvin Cycle. | glycolate-2-phosphate. This is | | | converted into GP in a reaction | | | that uses products of the Calvin | | | cycle, and ATP and releases | | | carbon dioxide. Because it uses | | | oxygen and releases CO~2~ it is | | | known as **photorespiration**. | +-----------------------------------+-----------------------------------+ Fortunately, RUBISCO has an affinity for CO~2~ that is 80 times higher than its affinity for oxygen, but in spite of this, photorespiration wastes both carbon and energy. About 25% of the products of the Calvin cycle are lost in photorespiration. So in many plants photosynthesis is 25% less efficient than it might be. All the evidence suggests that when RUBISCO evolved, the atmosphere was high in carbon dioxide with very little oxygen, so photorespiration never occurred and there was no selection pressure against it. Even today, with a high oxygen/low carbon dioxide atmosphere, photorespiration is not a problem for plants. There is no selection pressure for the enzyme to evolve to become more specific to carbon dioxide. A more efficient version of RUBISCO would, however, be extremely useful to people, because our crop plants could become 25% more productive.   **[Sequencing the Calvin Cycle]** Calvin used ^14^C to identify and sequence photosynthetic products. ^14^C is a radioactive isotope of carbon. It behaves as ^12^C would behave but, being radioactive, its presence can easily be detected. ^14^CO~2~ was fed to algal cultures which fixed the ^14^CO~2~ during photosynthesis in a special 'lollipop' vessel. Samples of algae were taken after varying periods of photosynthesis and the photosynthetic products identified. Calvin reasoned that the ^14^C label would appear first, and in the largest amount, in the first products of photosynthesis. The label would then be passed on to the subsequent products of photosynthesis in the order in which they were produced. The biochemical sequence he identified is now commonly referred to as the Calvin or C3 cycle. He separated and identified products of photosynthesis: - Paper chromatography to separate - Autoradiography to locate and identify (chromatograms placed on photographic film/paper. Radioactivity caused fogging when it was developed.) He produced graphs that clearly show the sequence of ^14^C fixation during photosynthesis. First product appears -- Glycerate 3-phosphate followed by - glyceraldehyde 3-phosphate followed by.... Note: sometimes the Calvin cycle is called the C3 cycle because the first stable product is glycerate-3-phosphate.