Dark reactions

  • 1,998 views
Uploaded on

Made for 2nd year Bioenergetics class

Made for 2nd year Bioenergetics class

More in: Technology , Business
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
No Downloads

Views

Total Views
1,998
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
0
Comments
2
Likes
1

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

  • 1. Photosynthesis: Calvin Cycle
  • 2. Photosynthesis grana disks 2 outertakes place in (thylakoids) membraneschloroplasts. stromaIt includes light compartmentreactions andreactions that arenot directlyenergized by light. ChloroplastLight reactions: Energy of light is conserved as  “high energy” phosphoanhydride bonds of ATP  reducing power of NADPH.Proteins & pigments responsible for the lightreactions are in thylakoid (grana disc)membranes.Light reaction pathways will be not be presented
  • 3. grana disks 2 outerCalvin Cycle, (thylakoids) membranesearlier designatedthe photosynthetic stroma"dark reactions," compartmentis now called thecarbon reactionspathway: ChloroplastThe free energy of cleavage of ~P bonds of ATP, andreducing power of NADPH, are used to fix andreduce CO2 to form carbohydrate.Enzymes & intermediates of the Calvin Cycle arelocated in the chloroplast stroma, a compartmentsomewhat analogous to the mitochondrial matrix.
  • 4. 2- H2C OPO 3 O C -O O C H C OH H C OH H C OH H2 C OPO 32- 2- H2C OPO 3 3-Phosphoglycerate Ribulose-1,5-bisphosphate (3PG) (RuBP)Ribulose Bisphosphate Carboxylase (RuBPCarboxylase), catalyzes CO2 fixation:ribulose-1,5-bisphosphate + CO2  2 3-phosphoglycerateBecause it can alternatively catalyze an oxygenasereaction, the enzyme is also called RuBPCarboxylase/Oxygenase (RuBisCO). It is the most
  • 5. H 2C OPO32 H 2C OPO32 H 2C OPO32 1 O C2 O C HO C CO2 CO2 O OH C OH C OH C O C 3H C OH H+ H C OH H C OH H2O H C OH 4 H 2C OPO32 H 2C OPO32 H 2C OPO32 H 2C OPO32 5ribulose-1,5- enediolate b-keto 3-phosphoglyceratebisphosphate intermediate intermediate (2) RuBP Carboxylase - postulated mechanism: Extraction of H+ from C3 of ribulose-1,5-bisphosphate promotes formation of an enediolate intermediate. Nucleophilic attack on CO2 leads to formation of a b-keto acid intermediate, that reacts with water and cleaves to form 2 molecules of 3-phosphoglycerate.
  • 6. 2 2 H2C OPO 3 H2C OPO 3 HO C CO 2 HO C CO 2 C O H C OH H C OH H C OH 2 2 H2C OPO 3 H2C OPO 3 Proposed b-keto acid 2-Carboxyarabinitol-1,5- intermediate bisphosphate (inhibitor)Transition state analogs of the postulated b-ketoacid intermediate bind tightly to RuBP Carboxylaseand inhibit its activity.Examples: 2-carboxyarabinitol-1,5-bisphosphate(CABP, above right) & carboxyarabinitol-1-phosphate (CA1P).
  • 7. RuBPCarboxylasein plants is acomplex(L8S8) of: RuBisCO PDB 1RCX RuBisCO PDB 1RCX 8 large catalytic subunits (L, 477 residues, blue, cyan) 8 small subunits (S, 123 residues, shown in red).Some bacteria contain only the large subunit, with thesmallest functional unit being a homodimer, L2.Roles of the small subunits have not been clearlydefined. There is some evidence that interactionsbetween large & small subunits may regulate
  • 8. PDB 1RCXLarge subunits within ribulose-1,5-RuBisCO are arranged as bisphosphateantiparallel dimers, with theN-terminal domain of onemonomer adjacent to the C-terminal domain of the other.Each active site is at aninterface betweenmonomers within a dimer,explaining the minimal 2L & 2S subunitsrequirement for a dimeric of RuBisCOstructure.The substrate binding site is at the mouth of an ab-barreldomain of the large subunit.Most active site residues are polar, including somecharged amino acids (e.g., Thr, Asn, Glu, Lys).
  • 9. O H Enz-Lys + NH3 + HCO3 Enz-Lys N C + H2O + H+ O Carbamate Formation with RuBP Carboxylase Activation"Active" RuBP Carboxylase has a carbamate thatbinds an essential Mg++ at the active site.The carbamate forms by reaction of HCO3 with thee-amino group of a lysine residue, in the presence ofMg++.HCO3 that reacts to form carbamate is distinct fromCO2 that binds to RuBP Carboxylase as substrate.Mg++ bridges between oxygen atoms of the carbamate& substrate CO .
  • 10. Binding of either RuBP or a transition state analogto RuBP Carboxylase causes a conformationalchange to a "closed" conformation in whichaccess of solvent water to the active site isblocked.RuBP Carboxylase (RuBisCO) can spontaneouslydeactivate by decarbamylation.In the absence of the carbamate group, RuBisCOtightly binds ribulose bisphosphate (RuBP) at theactive site as a “dead end” complex, with theclosed conformation, and is inactive in catalysis.In order for the carbamate to reform, the enzymemust undergo transition to the open conformation.
  • 11. RuBP Carboxylase Activase is an ATP hydrolyzing(ATPase) enzyme that causes a conformationalchange in RuBP Carboxylase from a closed to anopen state.This allows release of tightly bound RuBP or othersugar phosphate from the active site, and carbamateformation.Since photosynthetic light reactions produce ATP, theATP dependence of RuBisCO activation provides amechanism for light-dependent activation of theenzyme.The activase is a member of the AAA family ofATPases, many of which have chaperone-likeroles.RuBP Carboxylase Activase is a large multimeric
  • 12. Phosphoglycerate Glyceraldehyde-3-phosphate Kinase DehydrogenaseO O O OPO32 C ATP ADP C NADPH NADP+ CHOH C OH H C OH H C OH H2C OPO32 H2C OPO3 2 Pi H2C OPO3 2 3-phospho- 1,3-bisphospho- glyceraldehyde- glycerate glycerate 3-phosphate Glyceraldehyde-3-P Dehydrogenase catalyzes reduction of the carboxyl of 1,3-bisphosphoglycerate to an aldehyde, with release of Pi, yielding glyceraldehyde-3-P. This is like the Glycolysis enzyme running backward, but the chloroplast Glyceraldehyde-3-P Dehydrogenase uses NADPH as e donor, while the cytosolic Glycolysis enzyme uses NAD+ as e
  • 13. Continuing with Calvin Cycle:A portion of the glyceraldehyde-3-P is convertedback to ribulose-1,5-bisP, the substrate forRuBisCO, via reactions catalyzed by: Triose Phosphate Isomerase, Aldolase, Fructose Bisphosphatase, Sedoheptulose Bisphosphatase, Transketolase, Epimerase, Ribose Phosphate Isomerase, & Phosphoribulokinase.Many of these are similar to enzymes of Glycolysis,Gluconeogenesis or Pentose Phosphate Pathway,but are separate gene products found in thechloroplast stroma. (Enzymes of the other pathwayslisted are in the cytosol.)The process is similar to Pentose PhosphatePathway run backwards.
  • 14. Summary of Calvin cycle:3 5-C ribulose-1,5-bisP (total of 15 C) arecarboxylated (3 C added), cleaved,phosphorylated, reduced, & dephosphorylated,yielding6 3-C glyceraldehyde-3-P (total of 18 C). Ofthese: 1 3-C glyceraldehyde-3-P exits as product. 5 3-C glyceraldehyde-3-P (15 C) are recycled back into 3 5-C ribulose-1,5-bisphosphate. C3 + C3  C6 C3 + C6  C4 + C5 C3 + C4  C7 C3 + C7  C5 + C5
  • 15. Overall: TI glyceraldehyde-3-P dihydroxyacetone-P 5 C3  3 C5 AL, FBEnzymes: fructose-6-PTI, TKTriosephosphate Isomerase xyulose-5-P + erythrose-4-PAL, Aldolase AL, SBFB, Fructose-1,6- sedoheptulose-7-P TKbisphosphataseSB, xylulose-5-P + ribose-5-PSedoheptulose- EP IS (3) ribulose-5-PBisphosphatase PKTK, Transketolase (3) ribulose-1,5-bis-P
  • 16. CHO H C OHSummary of O C O H2C OPO32Calvin Cycle carbon glyceraldehyde- dioxide 3-phosphate3 CO2 + 9 ATP + 6 NADPH  glyceraldehyde-3-P + 9 ADP + 8 Pi + 6 NADP+Glyceraldehyde-3-P may be converted to otherCHO: • metabolites (e.g., fructose-6-P, glucose-1-P) • energy stores (e.g., sucrose, starch) • cell wall constituents (e.g., cellulose).Glyceraldehyde-3-P can also be utilized by plantcells as carbon source for synthesis of other
  • 17. grana disks 2 outer (thylakoids) membranes stroma compartment ChloroplastThere is evidence for multienzyme complexes ofCalvin Cycle enzymes within the chloroplast stroma.Positioning of many Calvin Cycle enzymes close tothe enzymes that produce their substrates or utilizetheir reaction products may increase efficiency ofthe pathway.
  • 18. Regulation of Calvin Cycle Regulation prevents the Calvin Cycle from being active in the dark, when it might function in a futile cycle with Glycolysis & Pentose Phosphate Pathway, wasting ATP & NADPH. Light activates, or dark inhibits, the Calvin Cycle (previously called the “dark reaction”) in several ways.
  • 19.  + H2O  OH + H  h stromaRegulation (alkaline) by Light. (acid inside Chloroplast thylakoid disks)Light-activated e transfer is linked to pumping of H+into thylakoid disks. pH in the stroma increases to about8.Alkaline pH activates stromal Calvin Cycle enzymesRuBP Carboxylase, Fructose-1,6-Bisphosphatase &Sedoheptulose Bisphosphatase.The light-activated H+ shift is countered by Mg++ releasefrom thylakoids to stroma. RuBP Carboxylase (instroma) requires Mg++ binding to carbamate at the active
  • 20. Some plants synthesize a transition-stateinhibitor, carboxyarabinitol-1-phosphate (CA1P),in the dark.RuBP Carboxylase Activase facilitates release ofCA1P from RuBP Carboxylase, when it isactivated under conditions of light by thioredoxin.
  • 21. Thioredoxin f PDB 1FAA disulfideThioredoxin is a small protein with a disulfide thatis reduced in chloroplasts via light-activated electrontransfer.
  • 22. ferredoxinRed ferredoxinOx thioredoxin thioredoxin S SH | S Ferredoxin- SH Thioredoxin ReductaseDuring illumination, the thioredoxin disulfide isreduced to a dithiol by ferredoxin, a constituent ofthe photosynthetic light reaction pathway, via anenzyme Ferredoxin-Thioredoxin Reductase.Reduced thioredoxin activates several CalvinCycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase,and RuBP Carboxylase Activase, by reducingdisulfides in those enzymes to thiols.
  • 23. PHOTORESPIRATION
  • 24.  Photorespiration occurs when the CO2 levels inside a leaf become low. This happens on hot dry days On hot dry days the plant is forced to close its stomata to prevent excess water loss. The plant continues fix CO2 when its stomata are closed, the CO2 will get used up and the O2 ratio in the leaf will increase relative to CO2 concentrations.
  • 25.  When the CO2 levels inside the leaf drop to around 50 ppm, Rubisco starts to combine O2 with RuBP instead of CO2 The net result of this is that instead of producing 2 3C PGA molecules, only one molecule of PGA is produced and a toxic 2C molecule called phosphoglycolateis produced.
  • 26. phosphoglycolate The plant must get rid of the phosphoglycolate since it is highly toxic. It converts the molecule to glycolic acid. The glycolic acid is then transported to the peroxisome and there converted to glycine.
  • 27. phosphoglycolate Glycolic acid In peroxisomes Glycine In mitochondria Serine
  • 28. • The serine is then used to make otherorganic molecules.• All these conversions cost the plantenergy and results in the net loss of CO2from the plant• To prevent this process, two specializedbiochemical additions have been evolvedin the plant world: C4 and CAMmetabolism.
  • 29. The C4 PATHWAY
  • 30. The C4 pathway is designed to efficiently fix CO2 at low concentrations and plants that use this pathway are known as C4 plants. These plants fix CO2 into a four carbon compound (C4) called oxaloacetate. This occurs in cells called mesophyll cells.
  • 31. 1. CO2 is fixed to a three-carbon compoundcalled phosphoenolpyruvate to produce thefour-carbon compound oxaloacetate. The enzyme catalyzing this reaction, PEPcarboxylase, fixes CO2 very efficiently so theC4 plants dont need to to have their stomataopen as much. The oxaloacetate is then converted toanother four-carbon compound called malatein a step requiring the reducing power ofNADPH
  • 32. 2. The malate then exits the mesophyllcells and enters the chloroplasts ofspecialized cells called bundle sheathcells. Here the four-carbon malate isdecarboxylated to produce CO2, a three-carbon compound called pyruvate, andNADPH. The CO2 combines with ribulosebisphosphate and goes through the Calvin
  • 33. 3.The pyruvate re-enters the mesophyllcells, reacts with ATP, and is convertedback to phosphoenolpyruvate, the startingcompound of the C4 cycle.
  • 34. The CAM PATHWAY
  • 35.  CAM plants live in very dry condition and, unlike other plants, open their stomata to fix CO2 only at night. Like C4 plants, the use PEP carboxylase to fix CO2, forming oxaloacetate. The oxaloacetate is converted to malate which is stored in cell vacuoles. During the day when the stomata are closed, CO2 is removed from the stored malate and enters the Calvin cycle
  • 36. Differences between calvin (C3) and C4 C3 C4 Temp 15-250 C  Temp 30-350 C Absence of malate  Presence of malate One carboxylation  2 carboxylation reactions reaction  HCO3 is the substrate CO2 is the substrate  Closed stomata to reduce water loss and Usual leaf structures concentrating CO2 in the bundle sheet cells  Additional ATP is required
  • 37. Comparison between C3, C4, and CAM C3 C4 CAM product G3P Malate Malate Day Day &night Night only &night Anatomy No bundle Bundle No bundle sheet cell sheet cell sheet cell No of stomata 2000- 10000- 100-800 31000 16000 Photorespirati Up to 40% Not Not on detectable detectable Species Wheat, Sugar cane Pineapple, rice, vanilla, cacti potato
  • 38. Factors affecting photorespiration O2: CO2Ratio If Cells Have Low O2 but Higher CO2, Normal photosynthesis i.e. Calvin Cycle Dominates C4Plants Have Little Photorespiration because They Carry the CO2to the bundle Sheath Cells and can Build up High [CO2]
  • 39. • Calvin Cycle Reactions always Favoredover Photorespiration• If Cells Have Higher O2and Lower CO2,Photorespiration Dominates• Temperature Photorespiration Increases withtemperature