Photosynthesis: Carbon Assimilation Processes

Photosynthesis (Carbon Assimilation) The light reactions result in the formation of the high-energy compounds ATP and NADPH. While these compounds can be used to drive metabolic processes, one additional critical reaction must occur: the fixation of carbon dioxide. Without CO2 fixation, the respiratory processes necessary to generate energy at night would result in an irreversible conversion of carbon compounds to CO2. In addition, carbon fixation is required to provide energy to non-photosynthetic tissues within the plant, and to supply the raw material required for growth of the plant. Animals lack the ability to perform net carbon dioxide fixation; reactions such as the pyruvate carboxylase reaction only result in temporary increases in the number of carbon atoms in the compound.7 Plants, however, have the capability of using the energy obtained from light to drive the permanent incorporation of carbon dioxide into structural molecules. The process for fixing and assimilating carbon is called the Calvin cycle in honor of the 1961 Nobel Laureate Melvin Calvin. Overview of the Calvin cycle The Calvin cycle begins with bisphosphate. Ribulose1,5-Bisphosphate Regeneration five-carbon Ribulose 1,5-Bisphosphate Ribulose 5-Phosphate Kinase Ribulose 5-Phosphate the carbohydrate Ribulose 1,5-Bisphosphate carboxylase/oxygenase (RuBisCO) ribulose-1,5- Carbon fixation CO2 ADP ATP 3-Phosphoglycerate (x2) ATP (x2) Phosphoglycerate kinase ADP (x2) 1,3-Bisphosphoglycerate (x2) NADPH (x2) Several Hexose Monosphosphate Pathway-like Reactions NADP (x2) Glyceraldehyde 3-Phosphate (x2) Glyceraldehyde 3-Phosphate Dehydrogenase Carbon assimilation Ribulose-1,5-bisphosphate acts as the carbon dioxide acceptor; following carbon dioxide addition, the resulting six-carbon compound is cleaved into two three-carbon 3-phosphoglycerate molecules. The 3-phosphoglycerate is then converted to 7 The pyruvate carboxylase reaction is the formation of the four-carbon oxaloacetate from the threecarbon pyruvate. Oxaloacetate may remain in the TCA cycle, or may be used for biosynthetic processes. When used for biosynthetic processes such as gluconeogenesis, the oxaloacetate loses the additional carbon. However, even carbon dioxide added to oxaloacetate that remains in the TCA cycle is effectively only fixed temporarily. Copyright © 2010-2011 by Mark Brandt, Ph.D. 21 glyceraldehyde-3-phosphate using energy obtained from the light reactions of photosynthesis. The glyceraldehyde-3-phosphate has several possible fates; however, the continuation of the carbon fixation process requires regenerating the ribulose-1,5-bisphosphate starting material. The cycle thus has three phases: carbon fixation, glyceraldehyde-3-phosphate formation and ribulose-1,5bisphosphate regeneration. Rubisco The rate-limiting enzyme in the Calvin cycle is ribulose 1,5-bisphosphate carboxylase/oxygenase (better known as rubisco). In most plants, rubisco is a complex of 8 large (53 kDa) and 8 small (14 kDa) subunits (
88). The large subunit is coded by a chloroplast gene, while the small subunit gene is located in the nucleus. In photosynthetic bacteria, rubisco is usually a dimer of proteins homologous to the plant large subunit. Rubisco is an intensively studied protein, because it is one of the most important enzymes in existence. Rubisco is present at a concentration of ~250 mg/ml in the chloroplast stroma, comprising ~50% of the total chloroplast protein. Plants production of rubisco exceeds that of any other enzyme, and probably any other protein, on earth. Current estimates suggest that the biosphere contains ~10 kg of rubisco per human (this works out to about 0.02 moles of the enzyme per person). This tremendous amount of protein is necessary because the catalytic efficiency of rubisco is fairly low: it has a kcat of 1 to 3 sec-1, and a rather low affinity for carbon dioxide. It is also necessary because rubisco catalyzes the wasteful oxygenase side reaction (discussed later); fixing sufficient carbon to support growth requires large amounts of this enzyme. The rubisco reaction is moderately complex. The general reaction mechanism is shown below. O O P O O O P O H2C O O C O O O P O H H2C O O C H + O C O O P O H2C O HO C C O C O O H2O O H2C O O P O H C OH H2C O HO C C C O O HO C OH H C OH C OH H C OH H C OH H C OH H C OH H2C O H2C O H2C O H2C O O O 3-Phosphoglycerate O C O O P O O P O O P O O P O H C OH O O O O H2C O Ribulose 1,5-bisphosphate Enediol intermediate
-Ketoacid intermediate O P O Hydrated intermediate O 3-Phosphoglycerate The enzyme has a basic residue that abstracts a proton from the 3-position carbon of the ribulose-1,5-bisphosphate substrate, producing the enzyme-bound ene-diol intermediate. The ene-diol acts as the carbon dioxide acceptor, with the carbon dioxide molecule forming a bond to the 2-position carbon. The six-carbon -ketoacid intermediate is then attacked by a water molecule, resulting in cleavage of the bond between the former 2-position and 3-position carbons of the ribulose-1,5- Copyright © 2010-2011 by Mark Brandt, Ph.D. 22 bisphosphate molecule. This results in the release of two 3-phosphoglycerate molecules (one of which, as released, contains a negatively charged carbon ion which then obtains a proton from the aqueous solvent). The overall reaction is therefore: Ribulose 1,5-bisphosphate + CO2 + H2O
2x 3-phosphoglycerate + 2 H+ The rubisco reaction thus yields two triose phosphate molecules from the single pentose bisphosphate. Note that ATP is not required for the rubisco reaction, a property that differs from most other carboxylase reactions; in this case, the reaction has a large negative
G° because of the release of the two triose phosphates. Carbon assimilation reactions The carbon-fixing rubisco reaction thus results in the release of 2 three-carbon 3phosphoglycerate molecules. The 3-phosphoglycerate molecules are converted to 1,3bisphosphoglycerate by phosphoglycerate kinase; this reaction requires ATP as a phosphate donor. The 1,3-bisphosphoglycerate is then converted to glyceraldehyde3-phosphate by glyceraldehyde-3-phosphate dehydrogenase. These two reactions use chloroplast isozymes of the glycolytic enzymes phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (note that the glyceraldehyde-3-phosphate dehydrogenase uses NADPH as a substrate rather than NADH). Because two 3-phosphoglycerate molecules are released by rubisco, two ATP and two NADPH are required to convert all of the triose phosphate to glyceraldehyde-3-phosphate. The ATP and NADPH required are produced by the light reactions, which therefore drive the entire process. O O P O H2C O H2O + CO2 2x 3-Phosphoglycerate 2 x ATP O O C O 2 x ADP C O H C OH H C OH H2C O O P O Rubisco H C OH CH2 O O P O O 2x NADPH 2 x NADP + 2 x Pi H C O C O Phosphoglycerate kinase H C OH CH2 O Glyceraldehyde3-phosphate dehydrogenase O P O O P O O O 2x 1,3-Bisphosphoglycerate O Ribulose 1,5-bisphosphate H C OH CH2 O O P O O 2x Glyceraldehyde 3-phosphate The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase reactions are reversible. In chloroplasts exposed to light and carbon dioxide, these reactions result in glyceraldehyde-3-phosphate formation due to the high concentrations of ATP, NADPH, and 3-phosphoglycerate. Copyright © 2010-2011 by Mark Brandt, Ph.D. 23 Regeneration of ribulose-1,5-bisphosphate Glyceraldehyde-3-phosphate can be converted to glucose without further input of energy. However, unless the ribulose 1,5-bisphosphate is regenerated the rubisco reaction will be unable to continue. The carbon assimilation process begins with three ribulose 1,5-bisphosphate (containing a total of 15 carbons); it ends with six glyceraldehyde-3-phosphate (containing a total of 18 carbons), a net gain of three carbons from carbon dioxide. Assuming that five triose phosphates are used to regenerate the starting ribulose 1,5-bisphosphate, the remaining triose phosphate can then be diverted to other processes. The regeneration process uses enzymes related to those of the hexose monophosphate pathway. Three-carbon units in the form of glyceraldehyde-3phosphate or dihydroxyacetone phosphate enter the pathway in a total of five places. The result of the pathway is the formation of three ribulose 1,5-bisphosphate molecules from the five trioses. 3+)#$&4%"#$%",(& )$#*&+,$& >".2=<&72/;(0&?( :%2;(".2=<&72/;(0&?( -.&'-./0( -)./0#$& 12%"#$%",(& &%)*&+,$& !"#$%&'( +,-.&'-./0( !"#$%"#%&'(#$& )$#*&+,$& 345 -)./0#$& 9.-&'( 12%"#$%",(& :,/0&'/01%( &%)*&+,$& 12%$%&'( +,-.&'-./0( 6"-5'4(%&0-'( 5(6&0(/%#7&'( )*8,-.'/0&'/01%( .(/&,(+%#0&'( )*'+,&'+,-%-'( =) 5(6&0(/%#7&'( 8,/0&'/01%( :%2;(?A>?B?;C= NAD NADH O NH3 H2C C H2C CH HO C O
-Hydroxyacid reductase HO C O NH3 H2C CH HO C O O O O Hydroxypyruvate Serine Serine DA>?B?;<= >?=0&'/0&@73$&71%( E,=0&'/0&@73$("1%( 5.&0&<('-"5H =0&'/0& !8+/9,(& (G&7/3"#H1%( !5! %"#$%",(& 6,+.#780,$& :);)',$& AK17&, 1$(%1%( =71'L&6('L1%1 J;<= <,0,(& :&"8:+#>&',$& F171%( =71'L&6('L1%1 C$?=%(,'.(/0.A;(%% <,0)64&'=8*& =3"#H1%( DJK FAG3>5H F171%( J;<= The pyruvate must be converted back to phosphoenolpyruvate using an unusual enzyme, pyruvate phosphate dikinase, which uses ATP to phosphorylate both the pyruvate and an inorganic phosphate (the pyrophosphate produced is then hydrolyzed to help drive the reaction). The C4 pathway thus requires the additional energy of two ATP equivalents per carbon fixed. However, the reduction in wasted energy due to lower oxygenase activity (and therefore lower photorespiration) allows C4 plants to outgrow C3 plants under conditions of high temperature with intense sunlight. C3 plants perform both photorespiration and the normal carbon-fixing Calvin cycle. C4 plants also perform both photorespiration and an identical carbon-fixing Calvin cycle. However, by adding an additional (energy-dependent) pathway, and by creating a spatial separation between the outside of the plant and the carbon fixation machinery, C4 plants perform much less oxygenase activity than do C3 plants, and therefore are more energy efficient under some conditions. In C3 plants, most photosynthesis occurs in the mesophyll cells. These cells are exposed to atmospheric oxygen and carbon dioxide. As a result, rubisco can use either carbon dioxide or oxygen as substrate, and therefore performs both carboxylase and oxygenase reactions. At high temperatures, rubisco has reduced affinity for carbon dioxide. Because the affinity for oxygen does not change Copyright © 2010-2011 by Mark Brandt, Ph.D. 33 significantly, the reduced affinity for carbon dioxide means that the oxygenase activity occurs more frequently at high temperatures. In C4 plants, the mesophyll cell fixes carbon dioxide temporarily by converting the three-carbon phosphoenolpyruvate to four-carbon oxaloacetate (C4 = four-carbon). This carbon is then transported to the bundle-sheath cell further inside the plant, where carbon dioxide is released. Because carbon dioxide concentration inside the bundle-sheath cell is higher, and because oxygen concentration is lower than in the mesophyll cell, the rubisco performs far fewer oxygenase reactions and more carboxylase reactions. This becomes important at high temperatures. D4?/71G% DE?/71G% 30M&'-.(<";ADJK A#%'.6(?&M?=71G% I('&-.2%% ;(%% ;F= B ==) =2= 345 30M&'-.(<";AJKA/?=ADJK A#%'.6(?&M?=71G% I('&-.2%% ;(%% AK3@(G1'( AK17&, 1$(%1%( -/.)$6# D1"-&K371'( F171%( =3"#H1%( DJK AK3@(G1'( -/.)$6# C$?=%(,L.(/0. ;(%% C$?=%(,L.(/0. D/<#&72%/'( ;(%% If the rubisco performs only carboxylase activity C4 plants require more energy per carbon fixed. However, if the C3 plant oxygenase/carboxylase ratio is high (e.g., at high temperatures), the energy saved by not performing the oxygenase pathway in C4 plants means that C4 plants may grow faster than C3 plants. Copyright © 2010-2011 by Mark Brandt, Ph.D. 34 Summary The light reactions of photosynthesis store energy in the form of ATP and NADPH. The reactions of the Calvin cycle (sometimes referred to by the historical but inaccurate title of the dark reactions) use ATP and NADPH to fix and assimilate carbon dioxide. The initial reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) which incorporates one molecule of carbon dioxide into the pentose and releases two 3-phosphoglycerate molecules. The 3-phosphoglycerate molecules are converted to glyceraldehyde-3-phosphate using chloroplast isozymes of the glycolytic enzymes phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Some of the glyceraldehyde-3-phosphate is diverted to other processes (such as starch synthesis, sucrose synthesis, or other forms of metabolism); the remainder is converted back to ribulose-1,5-bisphosphate by a series of reactions similar to those in the hexose monophosphate pathway. The Calvin cycle is stimulated by light, and is turned off in the dark, because light is required to generated the energy required for the carbon assimilation process. The rubisco enzyme catalyzes a wasteful side reaction using oxygen instead of carbon dioxide, resulting in the photorespiration pathway to regenerate the ribulose-1,5-bisphosphate substrate. C4 plants have evolved a somewhat more expensive variant of the Calvin cycle that reduces the oxygenase activity of rubisco. The overall process of photosynthesis uses the energy in light to synthesize carbohydrates, and, in plants, to release oxygen. These processes are required, directly or indirectly, for the survival of the vast majority of known life forms. Copyright © 2010-2011 by Mark Brandt, Ph.D. 35