Bacterial Metabolism and Energy Generation

1. Bacterial Metabolism and Energy Generation

2. An Overview of Metabolism

3. Metabolism – the sum of all chemical reactions occurring within a cell simultaneously. Involves degradation and biosynthesis of complex molecules. • Catabolism-the breakdown of larger, more complex molecules into smaller, simpler ones, during which energy is released, trapped, and made available for work • Anabolism-the synthesis of complex molecules from simpler ones during which energy is added as input

4. Multi-stage process of catabolism • Stage 1-breakdown of large molecules (polysaccharides, lipids, proteins) into their component constituents with the release of little (if any) energy

5. Stage 2-degradation of the products of stage 1 aerobically or anaerobically to even simpler molecules with the production of some ATP, NADH, and/or FADH2

6. Stage 3-complete aerobic oxidation of stage 2 products with the production of ATP, NADH, and FADH2; the latter two molecules are processed by electron transport to yield much of the ATP produced

7. Metabolic efficiency is maintained by the use of a few common catabolic pathways, each degrading many nutrients • Microorganisms are catabolically diverse, but are anabolically quite uniform • Amphibolic pathways function both catabolically and anabolically, and sometimes employ separate enzymes to catalyze the forward and reverse reactions; this separation enables independent regulation of the forward and reverse reactions

8. Definitions • Oxidation • Reduction • Redox reactions • Standard Reduction potential • Oxidative phosphorylation • Chemiosmosis • Electron transport chain

9. Oxidation • The loss of electrons from an atom or chemical compound. • Results in the generation of energy.

10. Reduction • The gain of electrons • Requires energy

11. Redox reactions • Reactions involving the transfer of electrons from a donor to an acceptor • = Redox couple • Reducing agent = reductant = donor • Oxidizing agent = oxidant = acceptor • Oxidant + ne- (number of electrons transferred) Reductant

12. Standard Reduction Potential (Equilibrium Constant = Eo’ at pH 7.0) • Measures the tendency of the reductant to lose electrons. • Redox couples with more negative Eo’ values will donate elecrron to redox couples with more positive Eo’ values, releasing free enegy (G) • Energy is required to reverse the process (e.g. photosynthesis)

13. The larger the difference in Eo’ values between electron donors and electron acceptors, the greater the free energy that is generated. • Therefore, the more negative the reduction potential, the better electron donor it is, and the more numerous the potential acceptors.

14. Oxidative phosphorylation • A metabolic sequence of reactions occurring within a membrane in which an electrons transferred from a reduced coenzyme by a series of electron carriers, establishing an electrochemical gradient across the membrane that drives the formation of ATP from ADP and inorganic phosphate by chemiosmosis. • Powered by redox reactions • Aerobic respiration uses O2 as TEA • Anaerobic respiration uses SO42-, NO3-, CO2 as TEA (not as efficient as using O2 as TEA)

15. Chemiosmosis • The generation of ATP by the movement of hydrogen ions into pores in the cytoplasmic membrane that are associated with the ATPase system.

16. Electron Transport Chain • A series of oxidation-reduction reactions in which electrons are transported from a substrate through a series of intermediate electron carriers to a final acceptor, establishing an electrochemical gradient across a membrane that results in the formation of ATP. • Electrochemical gradient = proton motive force • Examples of donors = coenzymes NADH, NADPH, and FADH2 --> often referred to as “reducing power”

17. Electron Transport and Oxidative Phosphorylation

18. Mitochondrial Electron Transport (Figure 9.13)

19. Three differences between prokaryotes and eukaryotes • #1: Prokaryotes use different electron carriers (cytochromes vary)

20. #2: The bacterial electron transport chain may be extensively branched with several terminal oxidases • Electrons may enter at several different points and use different TEAs • Often dependent on growing conditions of bacteria • Different cytochromes used depending on O2 status (log vs stationary)

21. #3: Electron transport in bacteria occurs in the cytoplasmic membrane • In eukaryotic cells this occurs on the inner membranes of mitochondria • But the mechanism of Ox-Phos is remarkably similar • Did mitochondria arise from bacteria? Endosymbiont theory

22. Electrons from NADH and FADH2 are transported in a series of redox reactions to a terminal electron acceptor • Reduced coenzymes (NADH and FADH2) generated in glycolysis and TCA cycles must be reoxidized

23. Oxidative Phosphorylation • Some of the energy liberated during electron transport is used to drive the synthesis of ATP in a process called oxidative phosphorylation

24. The chemiosmotic hypothesis of oxidative phosphorylation (Peter Mitchell) • Membrane-bound carriers transfer electrons to oxygen across a chain • Cytochromes, flavoprotein, quinones, non-heme iron containing proteins • Coupling of electron transport to oxidative phosphorylation

25. Postulates that the energy released during electron transport is used to establish a proton gradient (proton motive force due to different charge distributions) • As electrochemical potential is formed (PMF) protons are attracted back into the cell through a proton channel • F1F0 adenosine triphosphatase (F1F0 ATPase) in proton channel releases energy as protons enter

26. Blockers • inhibit the flow of electrons through the system • Examples: Cyanide or Azide (block electron transport between cyt a and O2 • Examples: Piericidin (competes with Coenzyme Q) and Antimycin A (blocks electron transport between cyt b and cyt c)

27. This proton-motive force is then used to drive ATP synthesis • Energy is converted to chemical energy by phosphorylation of ADP to ATP • High energy phosphate bonds used for biosynthetic pathways • Net result: • 1 NADP or NADPH = 3 ATP • 1 FADH2 = 2 ATP

28. Inhibition of aerobic synthesis of ATP • Inhibitors of ATP synthesis fall into two main categories:

29. Uncouplers • allow electron flow, but disconnect it from oxidative phosphorylation (inhibit ATP synthesis, not electron transport) • Uncouple electron transport from Ox-Phos  The energy from electron transport is lost as heat, not ATP • Examples: Valinomycin, Dinitrophenol (DNP)

30. The Yield of ATP in Glycolysis and Aerobic Respiration • The yield of ATP in glycolysis and aerobic respiration varies with each organism, but has a theoretical maximum of 38 molecules of ATP per molecule of glucose catabolized • Anaerobic organisms using glycolysis can only produce two molecules of ATP per molecule of glucose catabolized

31. Pasteur Effect • Switch from fermentation (anaerobic) to aerobic respiration when oxygen is available • Much more efficient • More ATP using oxygen as TEA • Sugar catabolism dramatically decreases

32. Distinction between Respiration and Fermentation • Respiration: • Electrons from oxidative process enter the electron transport system, protons are generated and energy is generated through OX-PHOS • Electrons are passed to an inorganic acceptor • Electron transport is used • OX-PHOS is used

33. Fermentation • Electrons and protons from oxidized substrates are transferred directly to another organic compound (organic acceptor) in the pathway • No electron transport • No OX-PHOS • Substrate-level phosphorylation used instead • The oxidation of a phosphorylated compound resuluting in the direct formation of a high-energy phosphate bond • Transferred to ADP to form ATP

34. Example of fermentation: • Glycolysis: • Glucose  Pyruvate  Lactic Acid or Ethanol • The final electron acceptor = pyruvate (or a product of pyruvate such as acetaldehyde intermediate) • Summary: In fermentation, organic compounds are the electron donors AND acceptors with some energy generated

35. The Breakdown of Glucose to Pyruvate

36. The glycolytic (Embden-Meyerhof) pathway is the most common pathway and is divided into two parts: • The 6-carbon sugar stage involves the phosphorylation of glucose twice to yield fructose 1,6-bisphosphate • requires the expenditure of two molecules of ATP

37. The 3-carbon sugar stage cleaves fructose 1,6-bisphosphate into two 3-carbon molecules, which are each processed to pyruvate • two molecules of ATP are produced by substrate-level phosphorylation from each of the 3-carbon molecules for a net yield of two molecules of ATP • 2 molecules of NADH are also produced per glucose molecule

38. 1 Glucose  2 Pyruvate • 2 ATP used • 4 ATP generated • Net yield = 2 ATP • 2 NAD used • 2 NADH generated • Net yield = 2 NADH (THIS MUST BE OXIDIZED TO KEEP THIS PATHWAY RUNNING.)

39. The pentose phosphate (hexose monophosphate) pathway uses a different set of reactions to produce a variety of 3-, 4-, 5-, 6-, and 7-carbon sugar phosphates • These phosphates can be used to produce ATP and NADPH, as well as to provide the carbon skeletons for the synthesis of amino acids, nucleic acids, and other macromolecules

40. The NADPH can be used to provide electrons for biosynthetic processes or can be converted to NADH to yield additional ATP through the electron transport chain

41. The Entner-Doudoroff pathway can also be used to produce pyruvate with a lower yield of ATP, but is accompanied by the production of NADPH as well as NADH.

42. Fermentation

43. In the absence of oxygen, NADH is not usually oxidized by the electron transport chain because no external electron acceptor is available • However, NADH must still be oxidized to replenish the supply of NAD+ for use in glycolysis

44. Fermentations are reactions that regenerate NAD+ from NADH in the absence of oxygen • Fermentations involve pyruvate or pyruvate derivatives as electron acceptors • Fermentations may or may not produce additional ATP for the cell

45. Six Common Pathways of Fermentation • Homoloactic acid • Alcoholoic • Propionic acid • Butylene glycol (Butanediol) • Mixed acid fermentation • Butyric acid, butanol, acetone

46. #1: Homolactic acid fermentation • Pyruvate  lactic acid • #2: Alcoholic fermentation • Pyruvate  acetaldehyde  ethanol

47. #3: Propionic acid fermentation • Pyruvate  acetic acid, OAA, malate, fumarate, succinate, propionate • #4: Butylene glycol fermentation (butanediol) • Pyruvate  acetolactic acid  acetoin  2,3 butylene glycol (2,3 butanediol)

48. #5:Mixed acid fermentation • Pyruvate  Lactate, formate, acetate, ethanol, succinate • #6: Butyric acid, butanol, acetone fermentation • Pyruvate  acetyl CoA, adetate, ethanol • Pyruvate  acetyl CoA, acetone, isopropanol • Pyruvate  acetyl CoA, butyryl CoA, butanol, butyric acid

49. Mixed Acid versus Butanediol Fermenters: 3 Tests to Divide Groups • Voges-Proskauer Test • Detection of acetoin (intermediary metabolite) • Methyl red test • Mixed acid fermenter produces a lot of acid • pH is ~4.4 • Red color produced for MAF only • CO2/H2 ratios • Mixed acid ~1:1 • Butanediol ~5:1

50. Respiration • Use of electron transport chain passing electrons to an inorganic terminal electron acceptor (TEA) • Energy generated through OX-PHOS • More energy efficient than fermentation