Carbon Fixation: The Calvin cycle, light regulation of carbon fixation, photorespiration in C4 and CAM plants Bioc 460 Spring Lecture 32 (Miesfeld) Sugarcane plants use C4 carbon fixation to limit photorespiration Melvin Calvin won a 1961 Nobel Prize for discovering carbon fixation Ribulose 1,5-bisphosphate carboxylase (Rubisco) is the most abundant enzyme on planet Earth

The photosynthetic electron transport chain operates in the light to generate chemical energy for use in the carbon fixation reactions of the Calvin cycle. The Calvin cycle enzyme Rubisco carboxylates ribulose bisphosphate (RUBP) to form a C-6 intermediate that is rapidly cleaved to form two moles 3- phosphoglycerate; three turns of the cycle are needed to generate one mole of glyceraldehyde-3P (GAP) from three moles of CO 2. Light activates enzymes in the Calvin cycle by two primary mechanisms, 1) increased Rubisco activity in response to elevated pH and Mg2+ in the stroma, and 2) thioredoxin-mediated reduction of disulfide bonds. Photorespiration is a wasteful side reaction of Rubisco that uses O 2 to generate 2-phosphoglycerate which must be metabolized in peroxisomes. The C4 and CAM carbon fixation pathways minimize the effects of photorespiration by increasing the local concentratation of CO 2 in the chloroplast stroma. Key Concepts in Carbon Fixation

Three stages of the Calvin Cycle: Fixation, Reduction, and Regeneration Plants store light energy in the form of carbohydrate, primarily starch and sucrose. The carbon and oxygen for this process comes from CO 2, and the energy for the energy for carbon fixation is derived from the ATP and NADPH made during photosynthesis. The conversion of CO 2 to carbohydrate is called the Calvin Cycle and is named after Melvin Calvin who discovered it. The Calvin Cycle requires the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase commonly called rubisco. The Calvin cycle generates the triose phosphates 3-phosphoglycerate, glyceraldehyde-3P (GAP) and dihydroxyacetone phosphate, all of which are used to synthesize the hexose phosphates fructose-1,6- bisphosphate and fructose 6-phosphate.

Hexose phosphates produced by the Calvin Cycle are converted to: 1) sucrose for transport to other plant tissues 2) starch for energy stores within the cell 3) cellulose for cell wall synthesis 4) pentose phosphates for metabolic intermediates

Basic scheme of the Calvin Cycle Three turns of the cycle results in the fixation of three molecules of CO 2 Key reaction in stage 1, catalyzed by rubisco enzyme, combines three molecules of ribulose-1,5- bisphosphate (RuBP), a five carbon (C5) compound, with three molecules of CO 2 to form six molecules of the C 3 compound 3- phosphoglycerate

Basic scheme of the Calvin Cycle The Calvin Cycle is sometimes called the Dark Reactions, but do not be fooled by this name - the Calvin Cycle is the most active during the daylight hours when ATP and NADPH are plentiful. The net reaction of three turns of the Calvin cycle can be written as: 3 CO RuBP + 6 NADPH + 9 ATP + 6 H 2 O → 1 GAP + 3 RuBP + 6 NADP ADP + 9 Pi If we just look at the fate of the carbons coming from CO 2 (C 1 ) in this reaction, we see that one net C 3 compound (glyceraldehydes-3P) is formed and three C 5 molecules (RuBP) are regenerated: 3 C C 5 → 1 C C 5 Why property of Calvin Cycle reactions make them Dark Reactions?

Stage 1: Fixation of CO 2 to form 3-phosphoglycerate To identify the metabolic intermediates in this process, Calvin and his colleagues used radioactive labeling with 14 CO 2 to follow carbon fixation in photosynthetic algae cells grown in culture. They found that within a few seconds of adding 14 CO 2 to the culture, the cells accumulated 14 C-labeled 3-phosphoglycerate, suggesting that this was the first product of the carboxylation reaction. Within a minute of adding 14 CO 2 to the culture, they found numerous compounds were labeled with 14 C, many of which were later identified as Calvin Cycle intermediates.

Rubisco reaction can be broken down into four basic steps 1)formation of an enediolate intermediate of RuBP 2)carboxylation by nucleophilic attack on the CO 2 3)hydration of 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate 4)aldol cleavage to form two molecules of 3-phosphoglycerate The rubisco reaction is very exergonic (ΔG°' = kJ/mol), with the aldol cleavage step being a major contributor to the favorable change in free energy

Rubisco is a multisubunit enzyme consisting of eight identical catalytic subunits at the core surrounded by eight smaller subunits that function to stabilize the complex and presumably enhance enzyme activity. Considering that rubisco plays a central role in all photosynthetic autotrophic organisms on earth, of which ~85% are photosynthetic plants and microorganisms that inhabit the oceans, rubisco is the most abundant enzyme on this planet.

Stage 2: Reduction of 3-phosphoglycerate to form hexose sugars 3-phosphoglycerate (product of the rubisco reaction) is converted to glyceraldehyde-3- phosphate (GAP) by two isozymes of phosphoglycerate kinase and glyceraldehyde-3P dehydrogenase. It is these two reactions that use the ATP and NADPH made during the light reactions. cleavage.

Stage 2: Reduction of 3-phosphoglycerate to form hexose sugars Remember that for every 3 CO 2 that are fixed by carboxylation of 3 RuBP molecules, six moles of 3- phosphoglycerate are generated by aldol cleavage. Therefore, 6 ATP and 6 NADPH are required for every 3 CO 2 that are converted to one net glyceraldehyde-3P. An additional 3 ATP are used in stage 3 to regenerate these 3 RuBP molecules. 3 CO RuBP + 6 NADPH + 9 ATP + 6 H 2 O → 1 GAP + 3 RuBP + 6 NADP ADP + 9 Pi

Stage 3: Regeneration of ribulose-1,5-bisphosphate In this final stage of the Calvin cycle, a series of enzyme reactions convert five C 3 molecules (GAP or DHAP) into three C 5 molecules (RuBP) to replenish supplies of this CO 2 acceptor molecule which is required in the rubisco reaction. This requires an additional 3 ATP. Two of the primary enzymes in this carbon shuffle are transketolase and transaldolase which are involved in interconverting C 3, C 4, C 6 and C 7 molecules:

“Carbon shuffle” reactions Five C 3 molecules of GAP are converted to three C 5 molecules consisting of two xylulose-5P and one ribose-5P. The two xylulose-5P and one ribose-5P molecules are first converted to three ribulose-5P molecules by the enzymes ribulose-5P epimerase and ribose-5P isomerase, respectively. The enzyme ribulose- 5P kinase catalyzes a phosphoryl transfer involving three ATP to generate the final three molecules of RuBP.

The net reaction of the Calvin Cycle can be broken down into two components 1) Synthesis of one glucose molecule from 6 CO 2 using 12 ATP and 12 NADPH 2) Regeneration of 6 RuBP using 6 ATP : Glucose synthesis 6 CO RuBP + 12 NADPH + 12 ATP + 10 H 2 O → 4 GAP + 2 DHAP + Fructose-6P + Glucose + 12 NADP ADP + 16 Pi Regeneration of RuBP 4 GAP + 2 DHAP + Fructose-6P + 6 ATP + 2 H 2 O → 6 RuBP + 6 ADP + 2 Pi Net reaction from six turns of the Calvin cycle 6 CO NADPH + 18 ATP + 12 H 2 O → Glucose + 12 NADP ADP + 18 Pi

Why must the Calvin Cycle be regulated? At night, plant cells rely on glycolysis and mitochondrial aerobic respiration to generate ATP for cellular processes. Since photophosphorylation and NADPH production by the photosynthetic electron transport system is shut down in the dark, it is crucial that the Calvin cycle only be active in the light. Otherwise, if glycolysis, the pentose phosphate pathway and the Calvin cycle were all active at the same time, then simultaneous starch degradation and carbohydrate biosynthesis would quickly deplete the ATP and NADPH pools in the stroma. Rubisco and several other enzymes are regulated by pH and Mg 2+, whereas, others are regulation by thioredoxin-mediated reduction of disulfide bonds.

In the absence of light, Calvin cycle enzymes have reduced activity and flux through the Calvin cycle is decreased dramatically. It makes sense that the enzymes would be inactive at times when ATP and NADPH levels are too low to support carbon fixation. Harvest moon over Tucson

When the sun comes up, light activation of the photosynthetic electron transport system causes stromal pH to increase from pH 7 to pH 8 as a result of proton pumping into the thylakoid lumen. This influx of H + into the lumen causes an efflux of Mg 2+ to the stroma to balance the charge. Rubisco and fructose-1,6- bisphosphatase (FBPase) activities are maximal under conditions of pH 8 and high Mg 2+. Sunrise hits the Old Pueblo

Activation by thioredoxin- mediated reduction of disulfide bridges Thioredoxin is a small protein of 12 kDa that is found throughout nature and functions as a redox protein that can interconvert disulfide bridges and sulfhydrals in cysteine residues of target proteins. As long as reduced thioredoxin is present in the stroma, these Calvin Cycle enzymes are maintained in the active state. However, when the sun goes down, spontaneous oxidation leads to their inactivation.

Photorespiration and Rubisco Rubisco also catalyzes a oxygenase reaction that combines RuBP with O 2 to generate one molecule of 3-phosphoglycerate (C 3 ) and one molecule of 2-phosphoglycolate (C 2 ). It is thought that this "wasteful" reaction belies the ancient history of the rubisco enzyme which has been around since before O 2 levels in the atmosphere were as high as they are today.

Photorespiration and Rubisco In order to salvage the carbon “wasted” on 2- phosphoglycolate, it must first be converted to glycolate, which is exported to peroxisomes to make glyoxylate and glycine which is then exported to mitochondria where two molecules of glycine are converted to one molecule of serine.

Photorespiration and Rubisco Oxygenation of RuBP, and metabolism of 2- phosphoglycolate by the glycolate pathway, is collectively called photorespiration because O 2 is consumed and CO 2 is released. However, unlike mitochondrial respiration, photorespiration requires energy input and is therefore considered by some to be a “wasteful” pathway in photosynthetic cells.

Rubisco is a carboxylase and an oxygenase

C 4 and CAM Carbon Fixation Pathways Plants in hot, sunny, climates are especially susceptible to photorespiration due to high O 2 :CO 2 ratios under these conditions. Oxygen is more soluble at high temperatures and this raises the O 2 :CO 2 ratio causing more photorespiration (O 2 competes with CO 2 for the rubisco active site). In the 1960s, Marshall Hatch and Roger Slack, plant biochemists at the Colonial Sugar Refining Company in Brisbane, Australia, used 14 CO 2 labeling experiments to determine what the initial products were in the carbon fixation reactions of sugarcane plants. To their surprise, they found that malate was more quickly labeled with 14 C than was 3-phosphoglycerate. Follow up work showed that plants such as sugarcane and corn, and weeds like crabgrass, thrive under high temperature conditions by having very low levels of photorespiration. The mechanism involves the carboxylation of phosphoenolpyruvate (PEP) by the enzyme PEP carboxylase to form oxaloacetate (OAA), a four carbon (C 4 ) intermediate that serves as a transient CO 2 carrier molecule.

Two variations of the "Hatch-Slack" pathway The C 4 pathway in tropical plants such as sugarcane that utilize two separate cell types to reduce photorespiration during the day. The CAM pathway found in desert succulents such as the giant saguaro cactus which captures CO 2 at night in the form of malate and releases during the day in the same cell.

C 4 Pathway in Sugarcane Mesophyll cells are responsible for CO 2 capture Interior bundle sheath cells (further away from atmospheric O 2 ), use CO 2 released from the C 4 intermediate malate to carry out the Calvin cycle reactions. This "separation in space" between the two cell types essentially eliminates the oxygenase reaction in rubisco and thereby blocks photorespiration. Note that two high energy phosphate bonds are required to convert pyruvate to phosphoenolpyruvate in mesophyll cells (PPi --> 2 Pi).

The additional input of energy required to temporarily store the CO 2 would seem to put C 4 plants at a disadvantage. However, the metabolic cost is more than compensated for by the increased carboxylation efficiency of rubisco in these plants once temperatures reach ºC and the O 2 :CO 2 ratio rises. In fact, considering that photorespiration in C 3 plants is a significant problem at high ambient temperatures, C 4 plants have a slight advantage under these conditions because the cost of two additional ATP to store CO 2, is slightly less than the one ATP and one NADPH needed to recycle 2-phosphoglycerate in the glycolate pathway (photorespiration). A special crabgrass puller outer tool! Crabgrass has the advantage here.

This growth advantage of C 4 plants at high temperatures is evident in the heat of summer where crabgrass, a C 4 plant, is able to invade a turf lawn consisting of C 3 grasses that are growth-inhibited by high rates of photorespiration. However, at more moderate temperatures in the spring when photorespiration rates are low in C 3 plants, the higher energy cost of the C 4 pathway in the crabgrass is a disadvantage and the turf grass is able to prevail.

CAM Pathway First discovered in succulent plants of the Crassulaceae family therefore called Crassulacean Acid Metabolism (CAM) pathway. The CAM pathway functions to concentrate CO 2 levels in the chloroplast stroma to limit the oxygenase activity of rubisco. CAM plants like the saguaro cactus use a temporal separation (Separation in Time)

CAM Pathway During the night when the stomata are open, CO 2 is captured by the mesophyll cells and incorporated into OAA by PEP carboxylase OAA is then reduced by the enzyme NAD- malate dehydrogenase to form malate. During the day, the CO 2 is released, allowing the Calvin Cycle to fix the CO 2 into carbohydrate.