CHEM-E3215 Advanced Biochemistry

Transcription

1 CHEM-E3215 Advanced Biochemistry 30. Jan Prof. Silvan Scheller Lecture 10 Energy conservation general (some calculations) Energy conservation in anaerobes: e.g. methanogensis Life close to the thermodynamic limit Electron bifurcation Electromicrobiology Details for project work 1

2 ΔG: The Gibbs Energy (free energy) A + B Substrates C + D Products Free Energy A + B -ΔG Free Energy +ΔG C + D C + D A + B Progress of reaction ΔG <0 (negative) Exergonic reaction Can proceed (if TS not too high..) Progress of reaction ΔG > 0 (positive) Endergonic reaction Would proceed into other direction

3 Energy conservation (in metabolisms): Utilize exergonic catabolic reaction to store (conserve) the energy Reaction can be coupled to the generation of ATP (substrate level phosphorylation, SLP) Reaction can be coupled to the production of an ion gradient (ion pump) Similar concept: Electron bifurcation (later this lecture) à May be considered as 3 rd way of energy conservation

4 Biochemical Thermodynamics ΔG = ΔH TΔS change in free energy = change in enthalpy - Temperature x change in entropy A + B Substrates C + D Products G = G + RT ln [C][D] [A][B] At equilibrium ΔG=0 G = -RT ln [C][D] [A][B] R= gas constant T= absolute temperature [A] [B] = molar concentrations G = standard free energy of change G= free-energy of change The standard free energy change ( G ) When all reactants and products at standard concentrations (1M) are allowed to reach equilibrium under standard conditions (25 C and 1atm). Depends on the nature of the reactants and the products G ʹ=Free-energy change under biochemical standard condition at ph=7, unit activities, 25 C = 298 K

5 ATP (adenosine triphosphate) ATP is the principal short-term energy-storage compound of the cells Standard free energy change of ATP hydrolysis: ATP + H 2 O ADP + Pi G = kj/mol Coupling the hydrolysis of ATP to reactions with a positive free energy change makes the latter favourable à Calculation: How much is one mol of ATP worth in a cell? (a base) Adenosine AMP ADP ATP Adenine Ribose

6 ATP yield from glucose oxidation Reaction sequence ATP yield per glucose molecule Glycolysis: Conversion of glucose into pyruvate (in the cytoplasm) Phosphorylation of glucose 21 Phosphorylation of fructose 6-phosphate 21 Dephosphorylation of 2 molecules of 1,3-BPG 12 Dephosphorylation of 2 molecules of phosphoenolpyruvate 12 2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate Conversion of pyruvate into acetyl CoA (inside mitochondria) 2 molecules of NADH are formed Citric acid cycle (inside mitochondria) 2 molecules of adenosine triphosphate are formed from 2 molecules of succinyl CoA 12 6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, a-ketoglutarate, and malate 2 molecules of FADH 2 are formed in the oxidation of 2 molecules of succinate Oxidative phosphorylation (inside mitochondria) 2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the glycerol 3-phosphate shuttle) 13 2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP 15 2 molecules of FADH 2 formed in the citric acid cycle; each yields 1.5 molecules of ATP 13 6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP 115 ATP Glucose Pyruvate Acetyl CoA Citric acid cycle 8 e Glycolysis CO 2 2 e 2 CO 2 Figure 17.4 The link between glycolysis Oxidative phosphorylation Net Yield per Molecule of Glucose 130 Source: The ATP yield of oxidative phosphorylation is based on values given in P. C. Hinkle, M. A. Kumar, From Stryer 7 th edition ~ 30 ATP

7 Standard reduction potentials of biol. relevant reactions Table 18.1 Standard reduction potentials of some reactions Oxidant Reductant n E9 0 (V) Succinate 1 CO 2 a-ketoglutarate Acetate Acetaldehyde Ferredoxin (oxidized) Ferredoxin (reduced) H 1 H NAD 1 NADH 1 H NADP 1 NADPH 1 H Lipoate (oxidized) Lipoate (reduced) Glutathione (oxidized) Glutathione (reduced) FAD FADH Acetaldehyde Ethanol Pyruvate Lactate Fumarate Succinate Cytochrome b (13) Cytochrome b (12) Dehydroascorbate Ascorbate Ubiquinone (oxidized) Ubiquinone (reduced) Cytochrome c (13) Cytochrome c (12) Fe (13) Fe (12) / 2 O H 1 H 2 O à Calculation: How much energy from NADH (FADH 2 ) + O 2? Note: E9 0 is the standard oxidation reduction potential (ph 7, 258C) and n is the number of electrons transferred. E9 0 refers to the partial reaction written as Oxidant 1 e 2 S reductant From Stryer 7 th edition

8 C metabolism Aerobic (e.g. glycolysis) Anaerobic (e.g. methanogenesis) Thauer et al. Nature reviews in microbiology 2008, 579

9 Energy conservation in anaerobes Differences aerobic in anaerobic respiration: No oxygen available Often reaction close to the thermodynamic equilibrium Challenges for anaerobic microbes: How to generate less than one ATP per substrate? How to ensure a flat energy profile? à How to couple endergonic with exergonic steps Awesome review: Thauer et al. Bacteriological Reviews 1977,

10 Extra slide: Acetoclastic methanogens and ATP mm acetate needed for growth (à Conserves more energy, outcompetes Methanosaeta at high substrate concentrations) 7 70 μm acetate needed for growth (à Conserves less energy, grows slower, but outcompetes Methanosarcina at low substrate concentrations) à Niche differentiation! Welte et al. Biochim Biophys Acta. 2014, 1130; Jetten et al. FEMS Microbiol. Rev. 1992, 181

11 How hydrogenotrophic methanogens generate ATP H 2 CO H 2 à CH H 2 O H 2 Methyl-transferase pumps 2 Na + outside H 2 ATPase uses Na + (assumed 4Na + /ATP) à 0.5 ATP per CH 4 produced. H 2

12 Look at pathway and its energy profile ΔG (kj mol -1 ) CO 2 Formyl!MFR Formyl!H 4 MPT MFR H 4 MPT MFR H 2 CO H 2 à CH H 2 O -5 Methenyl!H 4 MPT ΔG = -131 kj mol -1 (1 bar H 2 ) ΔG = -40 kj mol -1 (0.1 mbar H 2 )! These numbers assume that H 2 is directly used for all reduction steps, which is not the case. (Calculated from: Thauer et al. Nat. rev. in microbiol. 2008, 579, Box1) Methylen!H 4 MPT CH 3!H 4 MPT CH 3!S!CoM CH 4 H!S!CoM H 4 MPT H!S!CoB CoM!S!S!CoB -55 H 2 H 2 H

13 Look at pathway and its energy profile ΔG (kj mol -1 ) CO 2 Formyl!MFR Formyl!H 4 MPT MFR H 4 MPT MFR H 2 Concentrations of intermediates?? -5 Methenyl!H 4 MPT à Pathway needs to have reasonable concentrations for all intermediates to be efficient. Diffusion Occupy active site of next enzymes Allowing for flux of metabolites Methylen!H 4 MPT CH 3!H 4 MPT CH 3!S!CoM CH 4 H!S!CoM H 4 MPT H!S!CoB CoM!S!S!CoB -55 H 2 H 2 H

14 Solution: For methanogens with cytochromes growing on H 2 Energy conservation in Methanosarcina barkeri growing on CO 2 and H 2. (Rare case, usually cytochrome-containing methanogens do CH 3 -X fermentation, no) The first and last steps are chemiosmotically coupled. Thauer et al. Nature reviews in microbiology 2008, 579

15 Hydrogenotrophic methanogens without cytochromes: First and last step also coupled (RPG effect) Gunsalus + Wolfe Biochem. Biophys. Res. Comm. 1977, 790

16 RPG effect: How should this work?? Chemiosmotic coupling not possible, because no cytochromes (and anyway cell- extract used). Somehow the first and the last step needs to be coupled. Gunsalus + Wolfe Biochem. Biophys. Res. Comm. 1977, 790

17 Methanogens without cytochromes How is the first and last steps are coupled? Proposal by R. K. Thauer: Reduction of CoM-S-S-CoB and of Fd together by 2 H 2 Catalysed by the hydrogenase (MvhADG) heterodisulphide reductase (HdrABC) complex à Flavin-based electron bifurcation! Proposed: Thauer et al. Nature reviews in microbiology 2008, 579 Biochemisty: Kaster et al. PNAS 2011, 2981

18 Flavin-based electron bifurcation X-ray: Wagner et al., Science 2017, 699

19 Flavin-based electron bifurcation Proposed electron-transfer pathway. Wagner et al., Science 2017, 699

20 Methanogens without cytochromes Energy conservation in methanogens without cytochromes growing on CO 2 and H 2. The first and last steps are coupled by flavin-based electron bifurcation (yellow). W. Buckel + R.K. Thauer Biochimica et Biophysica Acta 2013, 94

21 Other example of flavin-based electron bifurcation (yellow) Flavin-based electron bifurcation (yellow): The electron donor (H 2 ) is split into a stronger (Fd 2- ) and a weaker donor (NADH) electron donor. Rnf complex: The strong electron donor (Fd 2- ) is converted into the weaker donor NADH and energy is conserved by pumping one ion outside the cell. Although the elucidation of the mechanisms of energy conservation in acetogens is still in its infancy,. Genome analyses will certainly pave the way toward a better understanding of the energetics, biochemistry, and physiology of acetogens. (Müller Appl. Environ. Microbiol. 2003, 6345) Buckel + Thauer

22 Summary of energy conservation See lecture notes. Further reading: Origin of life: The Origin of Membrane Bioenergetics : Lane et al. Cell 2012, 1406 Origin of life: Early bioenergetic evolution : Sousa et al. Phil Trans R Soc B 2013, 368 Life at G close to 0: The Minimum Biological Energy Quantum : Müller + Hess Frontiers in Microbiology 2017, V8-A2019

23 Project presentation Friday Feb. 2 nd at 9.15 in D422 Presentation: keep it short, max 20 min (send slides afterwards) 1 Page summary for your colleagues Additional slides/data, texts, pictures, tables, further information etc. (send electronically)

24 Additional slides (not part of exam): Electromicrobiology Pfeffer et al. Nature 2012, 218 General review see: Shi et al. Nat. Rev. Microbiol. 2016, 651

25 Cable bacteria Pfeffer et al. Nature 2012, 218

26 Mechanism of electrical conductance Pirbadian + El Naggar Phys. Chem. Chem. Phys. 2012, 13802

27 Disputed mechanism of conductance: via aromatic aa Lovely Current Opinion in Electrochemistry 2017, 190

28 Possible applications of electromicrobiology Choi and Sang Biotechnol Biofuels 2016, 9:11

29 Possible applications of electromicrobiology Cheng et al. Environ. Sci. Technol. 2009, 3953

30 Possible applications of electromicrobiology Methane to electricity Or reverse! Figure by McGlynn et al. Nature 2015, 534 See also: Wegener et al. Nature 2015, 587 Scheller et al. Science 2016, 703

31 Possible applications of electromicrobiology Butane to electricity Or reverse! Laso-Pérez et al. Nature 2016, 396