Bioenergetics: Lecture of May 21, The thermodynamics of biological energy production

Transcription

1 Bioenergetics: Lecture of May 21, 2009 Introduction to bioenergetics. The thermodynamics of biological energy production Kinetic aspects of bioenergetic processes The molecular and cellular organization of bioenergetic systems Photosynthesis structure of the photosynthetic reaction center photosynthetic electron transfer plant photosystems CO 2 fixation photosynthetic efficiency Biofuels Ethanol Respiration and ATP synthesis Haber-Bosch process and biological nitrogen fixation

2 photosynthesis - the reaction center h Bchl 2 membrane Bchl Bph Q A Fe Q B Bchl -chlorophyll Bph - bacteriopheophytin Q - quinone

3 why is the back reaction (to (Bchl) 2+ ) so unfavorable? why is the B branch so much slower than the A branch?

4 the inverted region

5 organization of photosynthetic electron transfer system H + cytochrome c h membrane Q QH 2 H + photosynthetic reaction center cytochrome bc1

6 Quinone structures and redox properties lipid soluble carrier of electrons and protons R 1 O O R 1 R +e - +e - + 2H + R 1 OH R 2 R 3 O oxidized quinone (Q 10 ) R 2 R 3 O - semiquinone (can be protonated) R 2 R 3 OH hydroquinone Q A -only oxidized d and semiquinone i states; t Q B - undergoes all three redox states

7 some classes of herbicides are quinone analogs that bind tightly to Q B site, but are not reduced.

8 how do the quinones get out? Roszak et al. Science 302, 1969 (2003)

9 Replenishing electrons to RC + the cytochrome c - RC complex PDB ID 1L9J

10 complementary interactions assure specificity in interprotein electron transfer cyt c 2 RC

11 QH 2 oxidation in cytochrome bc 1 : the Q-cycle

12 Overall stoichiometry of cyclic electron transfer in photosynthetic bacteria: the RC takes up one H + from the cytoplasm per electron in cytochrome bc 1, one H + is taken up from the cytoplasm, but two are released to the periplasm, per electron. net: 2H + are translocated across the membrane per electron; this creates a p that is used to synthesize ATP, but cannot generate reducing equivalents for biosynthesis in a cyclic process

13 photosynthesis in plants H + Buchanan, Gruissem, Jones Biochemistry and Molecular Biology of Plants

14 plant photosystems (I and II)

15 Z-scheme for non-cyclic photosynthesis electron transfer

16 photosystem II (plant photosystems are depicted upside-down relative to bacterial RCs )

17 photosystem II Zouni et al Nature 409, 739 (2001) PDB ID 1FE1

18 Ferreira, Iverson et al. Science 303, 1831 (2004)

19 Cofactor arrangement in PSII P680 th ti f hl h ll i i di i h t P680 - the separation of chlorophyll rings in dimer is somewhat greater than in the RC; shows more monomeric character

20 Light harvesting antenna system in PSII

21 3 oxygen evolution during photosynthesis steady state O 2 evolving state dark adapted state Buchanan, Gruissem, Jones Biochemistry and Molecular Biology of Plants

22 Oxygen evolving center: tetra-manganese cluster Zouni et al Nature 409, 739 (2001) Ferreira, Iverson et al. Science 303, 1831 (2004) Guskov et al Nature Structural Biology 16, 334 (2009)

23 Photosystem II contains several lipid molecules within the structure Lipids are probably important for turnover of D1 polypeptide, which is recycled every 30 min in the cell

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25 RC core photosystem I Jordan et al., Nature 411, 909 (2000) PDB ID: 1JB0

26 organization of electron transfer centers in PSI

27 P700 - dimer of chlorophylls h ll

28 arrangement of antennae chlorophylls op in PS I

29 Dutton et al. Adv. Prot. Chem. 63 (2003)

30 Rubisco

31 Reactions Catalyzed by Rubisco All Rubisco Enzymes are not specific for CO 2 over O 2 Many RuBisCo-like enzymes sequences identified in soil bacteria and archaea: functions are unknown

32 CO 2 metabolism - needs to exchange between gas and solution phases: CO 2 (gas) CO 2 (aq) + H 2 O H 2 CO 3 HCO 3- + H + CO 2 (gas) CO 2 (aq) CO 2 (aq) + H 2 O G = kj/mole HCO 3- + H + G = kj/mole reversible hydration catalyzed by carbonic anhydrase M. thermophila carbonic anhydrase M. thermoautotrophicum CA

33 Biofuels Engineer cell metabolic pathways to produce alcohol: methanol, ethanol, propanol, butanol Cells need fuel/energy: best source is glucose Cellulose can be degraded to glucose by cellulases

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35 Degradation of Cellulose is the Rate limiting step Faster Cellulases More stable cellulases (last longer in incubator, and thus the total yield of degraded cellulase is greater) Microorganisms make cellulosomes which have several different types of cellulose degrading enzymes: Progressive degradation

36 Cellulases are processive enzymes

37 Clostridium thermocellum

38 Corn Stover Particles Pre-treatment: 190C for 15 min Pre-treatment changes susceptibility of cellulose to enzyme degradation

39 Utilization of forestland and agricultural land could produce 1.3 billion tons of dry biomass This would produce sufficient biofuels to displace 30 percent of domestic petroleum consumption

40 Ethanol Butanol Gasoline Energy content 84, , ,000 BTU/gal. Vapor pressure psi at 100F Automobile compatibility Max 10% 100% 100%

41 Biofuels Biofuels will be a big part of America s and the world s energy future. Biofuels Initiative: To make cellulosic ethanol cost competitive with gasoline by To replace 30 percent of current levels of gasoline consumption with biofuels by 2030 (or 30x30). Increased economic growth Economic opportunities for domestic, rural economies Decreased petroleum trade deficit Broad-based environmental benefits Reduced greenhouse gas emissions Reduced petroleum use in fuel production Improved national energy security Reduced reliance on foreign sources of energy Decreased threat of supply disruptions due to natural disasters, political instability, and price volatility

42 Metabolic Engineering Maximize the level of NAD available Cell has to tolerate working concentrations of substrate and products

43 Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways Atsumi, Liao Nature (2008) 351: 86-90

44 Engineered biochemical pathway to make alcohols from intermediates of the amino acid biosynthesis pathways Atsumi, Liao Nature (2008) 351: 86-90

45 Engineering Alcohol Dehydrogenase Cofactor Specificity for Isobutanol Production glucose 2 pyruvate Net reaction: 2 NADH CO 2 alss acetolactate 1 glucose + 2 NADPH + 2 NAD + NADPH ilvc 1 isobutanol + 2 NADP NADH + 2 CO 2 23dihydroxy 2,3-dihydroxy-isovalerateisovalerate ilvd -keto-isovalerate 66% of glucose carbon results in isobutanol 33% carbon lost to CO 2 Ehrlich Pathway (Isobutanol) CO 2 kivd isobutyraldehyde NADPH isobutanol yqhd As constructed the pathway has a cofactor imbalance To produce NADPH the cells have to spend energy/carbon resulting in lower yield. Changing the cofactor specificity of ilvc and yqhd would solve the imbalance.

46 Metabolic Engineering Maximize the level of NAD available Cell has tolerate working concentrations of substrate and products Project Goals Engineer NADH-dependent KARI (ilvc) and IDH (yqhd) enzymes. Deliver a NADH-dependent IDH with 50% of yqhd NADPHdependent isobutyraldehyde reduction activity by March 2009 Integrate the engineered enzymes into the isobutanol pathway. Optimize isobutanol production via strain development.

47 Clostridium thermocellum Can completely consume insoluble cellulose Produces ethanol Produces other products in addition to ethanol Cannot genetically modify Clostridium

48 Multi-stage biofuel production

49 Evolution of improved endoglucanases cellulosomes l Cellulosomal cellulases from anaerobic bacteria Clostridium thermocellum will be parents of recombinant endoglucanases (EGs) Successful optimization of EG and CBH activity enables Successful optimization of EG and CBH activity enables growth on cellulose

50 Thermostable Cellulases from SCHEMA MTIKEMPQPKTFGELKNL... KETSPIPQPKTFGPLGNL... KQASAIPQPKTYGPLKNL... WRRRGIPGPLGYPLVGSF... WIRKGVKGPRGLPFLGVI... FIRKGIKGPRGFPGIGML... WIRKGVKGPRGFPFFGVI... WIRKGVKGPRGFPFFGVI... WMRKGIKGPRGLPFFGII... WMRKGVKGPRGRPFVGVL... WRRRGVVGPMGFPVLGVF... REKIGLSGPEPHWFLGNL... REKIGLTGPEPHWFMGNL... RSSIGIPGPPVHWLWGNL... E KVSKYPKGPLPLPFIGNI E = C ij ij Parent 2 Parent 3 Parent 1 ug Glucose/ /ug Enzyme C therophilum H insolens T reesei HJPlus Temperature (C)

51 Directed Evolution of Cyt P450 BM3 to convert propane to propanol

52 Sugar polymer feedstocks

53 Cellulase synthase can be expressed in cyanobacteria Cellulase synthase can make noncrystalline cellulose Appears to also produce glucose in the media(?)