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Electron Transport II; Photosynthesis I

Electron Transport II; Photosynthesis I. Andy Howard Biochemistry Lectures 3 November 2010. Electron Transport, concluded; Photosynthesis. We’ll finish our discussion of mitochondrial electron transport Then we’ll discuss photosynthesis. The proton pump Proton Leaks

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Electron Transport II; Photosynthesis I

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  1. Electron Transport II; Photosynthesis I Andy HowardBiochemistry Lectures3 November 2010 ETS; photosynthesis I

  2. Electron Transport, concluded; Photosynthesis • We’ll finish our discussion of mitochondrial electron transport • Then we’ll discuss photosynthesis ETS; photosynthesis I

  3. The proton pump Proton Leaks Moving ATP to the cytosol Control Photosynthesis General principles Chlorophyll Light reactions Photosynthesis Dark Reactions RuBisCO Calvin Cycle C5 to C3 to C6 Regenerating C5’s Energy bookkeeping Sucrose & Starch Other C-fixation paths Geometry & control What we’ll discuss ETS; photosynthesis I

  4. Proton Leaks • If NADH oxidation is uncoupled physiologically from ATP synthesis, heat is produced • This happens on purpose in homeotherms, particularly infants and hibernating animals • Uncoupling caused by thermogenin (UCP1), a channel-making protein ETS; photosynthesis I

  5. Mechanism of thermogenin • External Signal (cold?)  • Norepinephrine binds to external receptor  • adenylyl cyclase activation  • (ATP -> cyclic AMP)  • activation of protein kinase A  • phosphorylation of triacylglycerol lipase  • release of fatty acids  • activation of thermogenin so it leaks protons ETS; photosynthesis I

  6. Moving ATP to the cytosol • Most ATP used in cytoplasm for anabolic reactions • Need a transporter to get it out:adenine nucleotide translocase • ATP,ADP uncomplexed to Mg2+ here • Net loss of charge of -1 in matrix results • This powers the translocation but slightly impoverishes the gradient • Need a phosphate transporter inward too:phosphate symport with H+ ETS; photosynthesis I

  7. P/O Ratio • Common descriptor for the number of ATP-synthetic reactions that can be accomplished per atom of oxygen that gets converted to water. • P/O for NAD-dependent reactions is 2.5 • 10 protons translocated per NADH • 1 ATP produced for 4 protons pushed back • So 10 / 4 = 2.5 ATP per NAD reaction • P/O for Q-dependent reactions is 1.5:Only 6 protons in per QH2: 6/4=1.5. ETS; photosynthesis I

  8. Oxidation of a 2n-carbon fatty acid yields (n-1) QH2,(n-1) NADH, and n acetyl CoA. Initiating the process costs 2 ATPs. Assume we can get 10 ATP per acetyl CoA. How much ATP can we get from oxidizing palmitate? 104 ATP 106 ATP 108 ATP 112 ATP Undeterminable given the data supplied iClicker quiz question ETS; photosynthesis I

  9. Answer to iClicker question • Palmitate is a C16 carboxylic acid. Therefore in the conditions of the problem, 2n = 16, n = 8, n-1 = 7. • Thus we get 7 QH2, 7 NADH,8 acetyl CoA produced by its oxidation • Thus we get 7*2.5 + 7 * 1.5 + 8 * 10 = 17.5 + 10.5 + 80 = 108 ATP produced • Starting the process costs 2 ATP, so the net result is 106 ATP gained ETS; photosynthesis I

  10. NADH Shuttle mechanism I:glycerol phosphate shuttle • Fig.14.18 • Cartoon courtesy Indiana State U. ETS; photosynthesis I

  11. NADH transport II:malate-aspartate shuttle • Fig. 14.19 • Cartoon courtesy Leeds dentistry ETS; photosynthesis I

  12. Other terminal electron acceptors, donors • Note that life precedes both the evolution of photosynthesis and the appearance of high [O2] in the atmosphere • Chemiautotrophic bacteria depend on oxidizing H2, NH4+, NO2-, H2S, S, or Fe2+ for energy production • E.coli can use fumarate as terminal acceptor ETS; photosynthesis I

  13. Oxidative damage • Production of radicals is essentially unavoidable when O2, H2O present and redox reactions occur • Particular problem is superoxide, O2-• • Obligate anaerobes lack protection against these species, which arise whenever oxygen is involved in redox reactions in aqueous media • Aerobic organisms find ways to detoxify superoxide and related species ETS; photosynthesis I

  14. Human CuZnSOD; PDB 2WYT32 kDa dimer, 1.0Å Superoxidedismutase • Variety of structural forms; all involve metal ions • This human SOD is Cu-Zn • Reaction is2O2-• + 2H+ H2O2 + O2 • Resulting peroxide is detoxified by catalase:2H2O2 2H2O + O2 Thermus catalasePDB 2V8T, 1.0Å67kDa dimer+2Mn 2+ ETS; photosynthesis I

  15. Photosynthesis • Definition:harvesting of light to generate energy • Happens in plants, photosynthetic bacteria • Photosynthetic reactions offer source for carbon fixation as well as energy in photoautotrophs • In higher plants these events happen in the thylakoid disks of chloroplasts ETS; photosynthesis I

  16. Light reactions • Electrons are promoted from the ground state to excited states upon absorption of a photon by a chromophore Drawing courtesy JohnsonCounty Community College ETS; photosynthesis I

  17. Chlorophyll • Heme-like chromophore with Mg2+ in the center • Absorbs strongly in red and blue; therefore it appears green www.steve.gb.com/science/photosynthesis.html ETS; photosynthesis I

  18. -carotene Other light-absorbing pigments • Chlorophyll in its various forms is not the only light-absorbing pigment in plants and photosynthetic bacteria • Accessory pigments / accessory proteins involved in resonant energy transfers to chlorophyll • Examples: carotenoids (particularly -carotene), phycoerythrin, phycocyanin ETS; photosynthesis I

  19. Accessory pigments in phycobilisomes • Water absorbs red light strongly • Shorter wavelengths (higher energies) are more penetrating • Aquatic plants need accessory pigments that absorb in the range that’s available • Energy absorbed by phycobiliproteins is transferred ultimately to ChlA by Förster resonances ETS; photosynthesis I

  20. Special pair • Two out of the large collection of chlorophyll molecules within a single photosystem that are responsible for giving up electrons rather than just getting electrons excited into higher-energy states • Pair of P680 molecules in photosystem II are the special pair in that case • Other chlorophyll molecules and antenna molecules absorb photons too; these transfer energy to the special pair ETS; photosynthesis I

  21. Photosystem II • Beginning of sequence of energy-generating pathways in the chloroplast or the bacterial membrane • Involves P680, a chlorophyll positioned so that its absorption max is at 680nm • Absorption maximum depends on chromophore’s specific structure and on modulation by neighboring protein species ETS; photosynthesis I

  22. Electron translocation in photosystem II • Two protons move across the thylakoid membrane for each electron promoted and transferred—plus two protons associated with the conversion of QH2 back to Q • This provide proton pumping capability like that in mitochondria • Difference: gradient is dependent only on pH difference, not electrical potential ETS; photosynthesis I

  23. Photosystem I • P700 is primary photon acceptor • Similar translocations of protons • Net reduction of NADP again • Non-cyclic: we need to re-oxidize ETS; photosynthesis I

  24. Net light reactions • NADPH produced • Eight protons pass across • Energy is, in principle, available from both sources, but NADPH is employed in anabolic reactions rather than as a source of ATP • Net ATP production per photon: unclear. Probably about 2. ETS; photosynthesis I

  25. Dark reactions • Series of ordinary chemical reactions • Powered by reducing power in NADPH • Anabolic • Some common features with pentose phosphate pathway ETS; photosynthesis I

  26. Dark reactions: overview • RuBisCO: condenses one molecule of CO2 with one molecule of ribulose 1,5-bisphosphate (RuBP) to form 2 molecules of 3-phosphoglycerate • Several reductions and interconversions starting with phosphoglycerate • Pathway is cyclic in that RuBP is regenerated for additional reactions ETS; photosynthesis I

  27. RuBisCO reaction RuBP • Condensation of ribulose 1,5-bisphosphate (RuBP) with CO2 to produce two molecules of 3-phosphoglycerate • Enzyme is ribulose1,5-bisphosphate carboxylase / oxygenase • Unwanted (?) side-reaction: • RuBP + O2 3-phosphoglycerate + 2-phosphoglycolate • No net carbon incorporation 3-phosphoglycerate ETS; photosynthesis I

  28. RuBisCO structure • L8S8 stoichiometryin higher plants:Mol.Wt. L=55kDa;Mol. Wt. S=12 kDa • TIM barrels in both • All (?) catalytic activity in L (large) subunit • L coded for by chloroplast gene • S by nuclear genome • Does S play a controlling role? PDB 1WDDOctamer of L8S8 unitsL2S2 shownfrom rice(cf. fig. 15.21) ETS; photosynthesis I

  29. The unwanted (?) side-reaction of RuBisCO • Secondary reaction isribulose 1,5-bisphosphate+ O23-phosphoglycerate +2-phosphoglycolate • Uses up oxygen rather than CO2 • No net carbon incorporation into organic molecules 2-phospho-glycolate ETS; photosynthesis I

  30. RuBisCO regulation • Plant growth closely associated with carboxylation / oxygenation ratio:Carboxylation high means fast growth • Easy way to alter that: grow plants in high CO2 • Difficult to do that without animal toxicity! • Expensive to put your cornfield in a plastic bubble(but not impossible) • Attempts to engineer proteins that don’t do oxygenation(or even that have improved C/O ratios)have failed ETS; photosynthesis I

  31. Could you win genetically? • Attempts to engineer proteins that don’t do oxygenation(or even that have improved CO2/O2 activity ratios) have failed • There are some plants whose RuBisCO has a better SC/O than that of others • Maybe O2 and CO2 bind in precisely the same way! ETS; photosynthesis I

  32. Subsequent dark reactions, I • Pair of 3-phosphoglycerate molecules enter reductive pathway toward bigger sugars: the Calvin cycle • Almost all of these reactions are found in other pathways: • Glycolysis (but backward) • Gluconeogenesis • Pentose phosphate pathway • It’s a cycle because ribulose bisphosphate will be recreated ETS; photosynthesis I

  33. Subsequent dark reactions, II (cf. fig. 15.18) • Three glycolysis / gluconeogenesis rxns: • GAPDH reaction:1,3-bisP-glycerate + NADPH + H+glyceraldehyde-3-phosphate + NADP + Pi • TIM required to convert G3P to DHAP • Aldolase makes fructose 1,6-bisphosphate • Some RuBP will later be recycled back in to provide input to subsequent condensations with CO2 ETS; photosynthesis I

  34. Calvin cycle: first reaction • Begins with ATP-dependent phosphorylation of 3-phosphoglycerate to make 1,3-bisphosphoglycerate via phophosphoglycerate kinase • Same reaction found in gluconeogenesis; reverse of glycolytic step • Enzyme is 3-layer sandwich PDB 1V6S86 kDa dimer Thermus thermophilus Monomer shown ETS; photosynthesis I

  35. 2nd Calvin-cyclereaction: GAPDH • NADPH-dependent reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate • As in gluconeogenesis, reverse of glycolytic reaction • GAPDH: typical NAD(P) dependent oxidoreductase PDB 1RM4297 kDa octamerdimer + monomer shownspinach ETS; photosynthesis I

  36. The fates ofglyceraldehyde-3-phosphate • The pathway divides three ways at this metabolite • One equivalent toward fructose 1,6-bisphosphate and gluconeogenesis • Two head toward pentose phosphate pathway, where a second bifurcation happens ETS; photosynthesis I

  37. C3 to C6 • TIM converts one molecule of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate • Glyc-3-P and DHAP condense to form fructose 1,6-bisphosphate in standard aldolase reaction • Fructose 1,6-bisphosphatase removes the 1-phosphate to make fructose 6-phosphate • All of this happens in gluconeogenesis ETS; photosynthesis I

  38. Photorespiration • 2-phosphoglycolate is the productof the RuBisCO oxygenation reaction • 2-P-glycolate is decarboxylated:2 2-P-glycolate  CO2 + 3-P-glycerate +Pi • The 3-P-glycerate can re-enter the Calvin cycle, but at the cost of some carbon • This lossy pathway is known as photorespiration ETS; photosynthesis I

  39. Transketolase • As we saw in the PPP, fructose-6-P can react with glyceraldehyde-3-P in a transketolase reaction to form xylulose-5-phosphate and erythrose-4-phosphate • K6 + A3  A4 + K5 • Typical TPP binding structure PDB 1ITZ 297 kDa octamerdimer+monomer shown maize ETS; photosynthesis I

  40. Fates of DHAP • Can participate in F-6-P production • Can condense with erythrose-4-P in an aldolase reaction to form sedoheptulose 1,7-bisphosphate (K3 + A4  K7) • This can be dephosphorylated at the 1-position to form sedoheptulose 7-P via sedoheptulose 1,7-bisphosphatase ETS; photosynthesis I

  41. The final Glyc3-P • It can condense with sedoheptulose 7-phosphate in another transketolase reaction to form xylulose-5-phosphate and ribose-5-phosphate:K7 + A3  A5 + K5 (fig. 15.19) • The ribose-5-phosphate is an endpoint but it can also be isomerized to ribulose-5-phosphate • Xylulose-5-phosphate can be epimerized to form ribulose-5-phosphate too ETS; photosynthesis I

  42. Activation ofribulose-5-phosphate • Phosphoribulokinase uses ATP as a phosphate source to convert ribulose-5-phosphate to RuBP • Enzyme is similar to adenylate kinase PDB 1A7J32 kDa monomer Rhodobacter sphaeroides ETS; photosynthesis I

  43. What is unique here? • Not much • Last reaction is specificto Calvin cycle • Others are found in gluconeogenesis or pentose phosphate pathway or both • In this direction these reactions require the NADPH and ATP derived from the light reactions of photosynthesis Melvin Calvinphoto courtesyNobelprize.org ETS; photosynthesis I

  44. Bookkeeping for dark reactions • Numbers given on fig.15.19 presuppose 3 input RuBP molecules per run of the cycle • This makes it easy to divide up the Glyceraldehyde 3-P later • Net reaction is:3 CO2 + 9ATP + 6 NADPH + 5 H2O  glyceraldehyde 3-P + 9ADP +8 Pi + 6 NADP+ ETS; photosynthesis I

  45. Cost of making Acetyl CoA • We get back 2 NADH, 2 ATP when we convert glyceraldehyde 3-P to acetyl CoA • Therefore acetyl CoA costs 9-2 = 7 ATP and 6-2=4 NAD(P)H • At 2.5 ATP per NAD, that total is 7 + 2.5 * 4 = 17 ATP required per acetyl CoA • When we oxidize acetyl CoA we get 10 ATP (see TCA-cycle lecture),so we’re 10/17 = 59% efficient ETS; photosynthesis I

  46. Carbohydrate storage in plants • Glyc3P is converted to glucose-6-P or glucose by gluconeogenesis • Glycogen is storage polysaccharide in bacteria, algae, some plants • Other plants make starch (amylose or amylopectin) from glucose-6-P • Pathway begins with conversion of glucose-6-P to glucose-1-P, catalyzed by phosphoglucomutase ETS; photosynthesis I

  47. Starch synthesis • Glucose 1-P activated with ATP, not UDP • -D-glucose 1-P + ATP  ADP-glucose + PPi • Reaction driven to the right by hydrolysis of PPi • ADP glucose is added to growing starch molecule with release of ADP:ADP-glucose + (Starch)nADP + (Starch)n+1 • Branching in amylopectin accomplished as in glycogen(Yao et al (2004) Plant Physiol.136:3515) ETS; photosynthesis I

  48. Diurnal variations in starch • Starch synthesis in daylight:ATP is readily available • Starch degradation at night • Starch phosphorylase cleaves starch to produce glucose-1-phosphate;glucose-1-P to triose phosphates by glycolysis • Enzyme is similar to glycogen phosphorylase • PLP-dependent PDB 2C4M 350 kDa tetramer Corynebacterium callunae ETS; photosynthesis I

  49. Alternative path for night-time starch degradation • Starch to dextrins via amylase • Dextrins are oligosaccharides beginning with a -1,6 link • Dextrins eventually degraded to glucose • Glucose is phosphorylated by hexokinase • Enzyme: sheet domain + TIM barrel PDB 1HT645 kDa monomer barley ETS; photosynthesis I

  50. Sucrose: mobile carbohydrate • Synthesized in chloroplast-containing cells; exported tovascular system so otherplant parts can use it • Two fructose 6-phosphatemolecules are starting points(fig 15.25) ETS; photosynthesis I

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