# Structural Biochemistry/Calvin Cycle
## Introduction

    Calvin Cycle is also known as the dark reaction part of the photosynthesis in which reduction of carbon atoms from carbon dioxide to a reduced state of hexose occurs by utilizing ATP and NADPH produced by the light reactions. Another reason why Calvin Cycle is known to be the dark reaction is because unlike light reactions, this reaction is independent of the presence of light. This cycle was first formed by Melvin Calvin. The Calvin Cycle uses sunlight as an energy source to synthesize glucose from carbon dioxide gas and water for photosynthetic organisms. This introduces all the carbon atoms used as a fuel source and as backbones of biomolecules in life. There are a lot of similarities between the Calvin Cycle and the Pentose Phosphate Pathway. Like mirror images of each other, the pentose phosphate pathway generates NADPH by breaking down the glucose into carbon dioxide. Similarly, the Calvin Cycle reduces the carbon dioxide to generate hexoses using NADPH.
Calvin Cycle Intermediate Biochemists tried to figure out the mechanism of carbon dioxide fixation, believing that agricultural photosynthesis could be made more efficient. In each "turn" of the cycle, one molecule of carbon dioxide is condensed with the five-carbon sugar. The resulting six-carbon intermediate splits into two molecules of 3-phosphoglycerate. Besides, the water and the phosphate group are recycled during biosynthetic assimilation of G3P [An Evolving Science].

## Stages of Calvin Cycle

The stages of Calvin Cycle occurs in the stroma of chloroplasts, the photosynthetic organelles.
Three stages include:
1) Two molecules of 3-phosphoglycerate formed by fixation of carbon dioxide by ribulose 1,5-bisphosphate
    In the beginning of this process, the ribulose 1,5-bisphosphate is converted into a highly reactive enediol intermediate. With the enediol intermediate, the carbon dioxide molecule is condensed into an unstable six-carbon compound. Rapidly, this unstable compound is hydrolyzed to two molecules of 3-phosphoglycerate. This reaction is highly exergonic with the Gibbs free energy equalling to -51.9 kJ/mol. This is catalyzed by rubisco which is also known as ribulose 1,5-bisphosphate carboxylase / oxygenase, an enzyme found in the stromal surface of the thylakoid membranes of chloroplasts. This reaction is very important because it is the rate-limiting step of the hexose synthesis. The structure of rubisco in chloroplasts contains eight large subunits (L, 55-kd) and eight small subunits (S, 13-kd). Each of the L subunits have a regulatory site and a catalytic site. Each of the S chains enhance L chains’ catalytic activities. Rubisco is known to be one of the most abundant enzymes and even the most abundant protein in the biosphere. Due to its slowness, rubisco must have large amounts present for the catalysis to work.
    - Rubisco: For activity, it requires a bound divalent metal ion, commonly magnesium ion. By stabilizing a negative charge, the magnesium ion serves to activate a bound substrate molecule. It requires a carbon dioxide molecule other than the substrate to conclude the assembly of the magnesium ion binding site in rubisco. This carbon dioxide molecule is added to the uncharged ε-amino group of lysine 201 which forms a carbamate. Then, the negatively charged adduct binds to the magnesium ion. Although the formation of the carbamate will form spontaneously at a lower rate, it is enabled by the enzyme rubisco activase. Magnesium ion plays an important role in binding ribulose 1,5-bisphosphate and activating it to react with carbon dioxide. Magnesium ion and ribulose 1,5-bisphosphate bind together through its keto and adjacent hydroxyl group. The complex forms an enediolate intermediate through deprotonation. This reactive species couples with carbon dioxide and forms a new carbon-carbon bond. Including the newly formed carboxylate, the product is coordinated to the magnesium ion through three groups. An intermediate is formed when H$_2$O is added to β-ketoacid which cleaves to form two molecules of 3-phosphoglycerate.
    - Rubisco also causes catalytic imperfection by catalyzing a wasteful oxygenase reaction. Instead of reacting with carbon dioxide, the magnesium ion sometimes reacts with O$_2$ which catalyzes a deleterious oxygenase reaction. The resulting products of this reaction are 3-phosphoglycerate and phosphoglycolate. Just like the carboxylase reaction, this oxygenase reaction requires the lysine 201 to be in the carbamate form. However, rubisco is prohibited from catalyzing the oxygenase reaction when carbon dioxide is not present because the carbamate only forms when carbon dioxide is present.
2) Hexose sugars formed by the reduction of 3-phosphoglycerate
The resulting product of rubisco, 3-phosphoglycerate, is converted into fructose 6-phosphate which isomerizes to glucose 1-phosphate and glucose 6-phosphate. Mixture of three phosphorylated hexoses is known as hexose monophosphate pool. The reaction of this conversion is very similar to the gluconeogenic pathway, except that glyceraldehyde 3-phosphate dehydrogenase is specific for NADPH rather than NADH which generates glyceraldehyde 3-phosphate (GAP). Carbon dioxide is brought up to the level of a hexose by the product catalyzed by rubisco and these reactions. Then, carbon dioxide is converted into a chemical fuel at the expense of NADPH and ATP which are generated from the light reactions.
3) Fixation of more carbon dioxide through the regeneration of ribulose 1,5-bisphosphate
The last phase of the Calvin Cycle is the regeneration of ribulose 1,5-bisphosphate, which is the acceptor of carbon dioxide in the first phase. From six-carbon and three-carbon sugars, a five-carbon sugar must be constructed. In the process of rearranging the carbon atoms, transketolase and aldolase play a major role. The transketolase transfesr a two-carbon unit from a ketose to an aldose by utilizing the coenzyme thiamine pyrophosphate (TPP). On the other hand, aldolase catalyzes an aldol condensation between an aldehyde and dihydroxyacetone phosphate (DHAP). Although this enzyme agrees with wide variety of aldehydes, it is very specific for dihydroxyacetone phosphates. In sum, when forming the five-carbon sugars, transketolase converts the three carbon and the six carbon sugars into a five carbon sugar and a four carbon sugar. The next process is when aldolase combines the four carbon sugar and a three carbon sugar to form a seven carbon sugar. The final step is that the seven carbon sugar reacts with another three-carbon sugar in order to form two more five carbon sugars. When the process for forming five carbon sugars are complete, ribose 5-phosphate is converted into ribulose 5-phosphate by the phosphopentose isomerase. Meanwhile, xylulose 5-phosphate is converted into ribulose 5-phosphate by phosphopentose epimerase and ribulose 5-phosphate is converted into ribulose 1,5-bisphosphate by phosphoribulose kinase. The following reaction shows the overall sum:
Fructose 6-phosphate + 2 glyceraldehyde 3-phosphate + dihydroxyacetone phosphate + 3 ATP → 3 ribulose 1,5-bisphosphate + 3 ADP
Calvin cycle requires six rounds to be completed since in each round, one carbon atom is reduced. In order to phosphorylate 12 molecules of 3-phosphoglycerate to 1,3-bisphosphoglycerate, 12 molecules of ATP are expended. In order to reduce 12 molecules of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate, 12 molecules are NADPH are consumed. This is the net reaction of the Calvin cycle:
6 CO$_2$ + 18 ATP + 12 NADPH + 12 H$_2$O → C$_6$H$_{12}$O$_6$ + 18 ADP + 18 P$_i$ + 12NADP$^+$ + 6H$^+$
Below shows a diagram of the net reaction of the Calvin cycle:
Roles of Hexose
In plants, there are two major storage forms of sugar which include starch and sucrose. Starch is very similar to its animal counterpart glycogen but has less branches since it has a smaller proportion of α-1,6-glycosidic linkages. Also, the activated precursor is ADP-glucose, not UDP-glucose. Starch is commonly known to be a polymer of glucose residues which is synthesized and stored in chloroplasts. Distinctly, sucrose, a disaccharide, is synthesized and stored in the cytoplasm. Plants are able to transport triose phosphates from the chloroplasts to the cytoplasm, but they lack the potential to transport hexose phosphates across the chloroplast membrane. In exchange for a phosphate through the phosphate translocator, the triose phosphate intermediates cross into the cytoplasm. From the triose phosphates, fructose 6-phosphate is formed which joins the glucose unit of UDP-glucose. This forms the sucrose 6-phosphate. The phosphate hydrolyzes and yields sucrose which is stored in many plant cells.

## Activation of Calvin Cycle

Regulation occurs when the stromal environment alters by the light reactions. pH increases in the light reactions and concentrations of magnesium ion, NADPH, and reduced ferredoxin. These changes help couple the Calvin cycle to the light reactions. Specifically, rubisco gets activated when the concentration of these molecules increases and the pH increases. Activity of rubisco increases because light creates the carbamate formation which is a necessity in enzyme activities. In the stroma, when the concentration of magnesium ion increases, the pH also increases from 7 to 8. From the thylakoid space, the magnesium ions are released in order to create the influx of protons into the stroma. Carbon dioxide is added to the rubisco’s deprotonated form of lysine 201 while magnesium ion is bound to the carbamate in order to generate enzyme’s active form. Therefore, the light generates the regulatory signals, ATP, and NADPH.
Thioredoxin
One of the important molecule in regulating the Calvin cycle is known as thioredoxin. When thioredoxin is oxidized, it contains a disulfide bond. This disulfide bond is converted into two free sulfhydryl groups when the thioredoxin is reduced with the reduced ferredoxin. Reduced form of thioredoxin can cleave disulfide bonds in enzymes which activates some of the Calvin cycle enzymes and inactivates some of the degradative enzymes. Examples of enzymes that are regulated by thioredoxin include: rubisco, fructose 1,6-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, sedoheptulose 1,7-bisphosphatase, glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, phosphoribulose kinase, and NADP$^+$-malate dehydrogenase.
C$_4$ Pathway
By having a high concentration of carbon dioxide at the site of the Calvin cycle, plants are able to prevent very high rates of wasteful photorespiration when growing in hot climates. The process behind this is that C$_4$ (four carbons) compounds carry carbon dioxide from mesophyll cells. Carbon dioxide is concentrated by the ATP in mesophyll cells in the bundle-sheath cells. This decarboxylation of C$_4$ compounds in the bundle-sheath cells have the ability to maintain high concentrations of carbon dioxide in the Calvin cycle. The remaining three carbons are returned to the mesophyll cell to proceed another round of carboxylation. The transportation of the carbon dioxide in the C$_4$ pathway begins inside the mesophyll cell when the carbon dioxide and phosphoenolpyruvate is condensed to form oxaloacetate. This reaction is catalyzed by the phosphoenolpyruvate carboxylase. At times, by an NADP$^+$ linked malate dehydrogenase, oxaloacetate may be converted into a malate. This malate enters the bundle-sheath cell and is decarboxylated inside the chloroplasts. By condensing the ribulose 1,5-bisphosphate, the released carbon dioxide enters the Calvin cycle. In the last process, pyruvate by pyruvate-P$_i$ dikinase forms the phosphoenolpyruvate. This is the C4 pathway net reaction:
CO$_2$ (mesophyll cell) + ATP + 2H$_2$O -> CO$_2$ (bundle-sheath cell) + AMP + 2 P$_i$ + 2 H$^+$
References
Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. Biochemistry. 7th ed. New York: W.H. Freeman, 2012. Print.