Metabolism. Fermentation vs. Respiration. End products of fermentations are waste products and not fully.

Transcription

1 Outline: Metabolism Part I: Fermentations Part II: Respiration Part III: Metabolic Diversity Learning objectives are: Learn about respiratory metabolism, ATP generation by respiration linked (oxidative) phosphorylation, Electron transport Fermentation vs. Respiration End products of fermentations are waste products and not fully. Still some useful left in the products How can a microorganism get more energy from glucose? 1

2 Use respiration and the tricarboxylic acid cycle TCA, citric acid cycle, Krebs cycle Aids in oxidizing to CO 2 Stores H+/electrons in reduced : NADH/H+ and FADH 2 One SLP step produces GTP (gets converted to ATP) Major pathway in aerobic respiration How many ATPs are produced? TCA NADH = 8 FADH = 2 GTP = 2 Cash em in 3 ATP : 1 NADH 2 ATP : FADH 1 ATP : GTP 1 Glucose ATP + 2 NADH (2 pyruvates) 2 2 Glycolysis/TCA 38 ATP per glucose 2

3 Fate of reduced coenzymes generated by TCA cycle they are oxidized by enzymes arranged in an electron transport chain. 2H+ 2H+ 2H+ NADH 2 2H+ NAD+ ADP 2H+ H+ ATP 2H + 1/2 O 2 H 2 O H+H+ H+ H+ H+H+ Chemiosmotic hypothesis: proton gradient is used to for chemical, osmotic, and mechanical work. Overview of Respiration 3

4 Electron transport and Oxidative Phosphorylation Electron transport & oxidative phosphorylation Required components: cytoplasmic redox reaction proton + charge gradient membrane-bound Electron transport phosphorylation occurs during: Respiration: An aerobic or anaerobic catabolic process. An organic or inorganic electron donor is oxidized using O 2 ( or an O 2 substitute) as the final electron acceptor Photosynthesis: Capture and use of Light Energy to fix [i.e. Incorporate] carbon into biomass. 4

5 What is the proton motive force (Δp)? p = Ψ -Z ph H+ H + H + develops Ψ = electrical l H + A - H + H + A - H + H H + + A - H A + - develops membrane potential, in mv ph = ph gradient Z = 2.3RT/F = 60 mv In - e- + Out develops Ways to make a proton motive force 1. : protons are pumped from inside the cell to the outside. Eg. NADH dehydrogenase and cytochrome c oxidase 2. : protons are transferred to quinones on CM inner leaflet and released on the outer leaflet : protons are consumed by reduction reactions on the inner leaflet of the CM Eg. O 2 reduced to H 2 O 5

6 Structure and function of ATP synthase (ATPase). Some of the energy liberated during electron transport is used to drive the synthesis of ATP in a process called oxidative phosphorylation h Uses the electrochemical proton gradient (part of the PMF) Can run in reverse to generate a proton gradient: 1 mol of F1 >> hydrolyzes 10 4 ATP to ADP + Pi 3-4 protons per ATP synthesized There is evidence for conformational changes and molecular rotation in the ATP synthase complex during proton movement across the membrane Redox reactions are essential to the function of electron transport chains e - In respiration the TEA is usually obtained from the external environment. The reduced TEA is usually secreted 6

7 Oxidation-Reduction Reactions and Electron Carriers Oxidation-reduction (redox) reactions involve the transfer of electrons from a donor (reducing agent or reductant) to an acceptor (oxidizing agent or oxidant) The equilibrium constant for the reaction is called the standard reduction potential (E 0 ) and is a measure of the tendency of the reducing agent to lose electrons Prokaryotes use electron carriers to transfer electrons from a reductant to an acceptor with a more positive (higher) reduction potential, and they thereby allow the release of free energy, which is often used in the formation of ATP. Biological cells have a variety of electron carriers, and each is used in particular types of redox reactions; the particular carrier used in any given reaction will depend on the nature and location of the reaction Quinone/Quinol Hydrophobic, non-protein molecules (Fig. 5.18) Accepts 2e- and 2H+ but only donates 2e- to next redox partner Q-loop or Q-cycle for proton translocation Many different types, ubiquinone, menaquinone 7

8 The Q-Loop and PMF Cytochromes In Shewanella CymA tetraheme cytochrome Contain iron porphyrin ring (heme) Fe2+ Fe3+ during oxidation. Electron transfer only (1 e-) Many different types, numbers of hemes, called cyt a, cyt b, cyt c CXXCH X = amino acid Motif for c-type cytochromes 8

9 CXXCH in the protein structure Cys His Cys Heme Iron-Sulfur Cluster Electron transfer only Exist as Fe-S clusters of different types Ferridoxin is an example of one. 9

10 Flavins Found in membrane proteins (integral or peripheral). Accepts electrons and protons from NADH Flavins only donate electrons Redox reactions & growth substrates 10

11 Redox reactions and reduction potentials (E o ) E o is the tendency of a substrate to donate or accept electrons given. Measured in Volts and determined under standard conditions: ph 7.0, 1 M, 25 C Electrons do not exist in solution so half reactions must be coupled to other ones The difference in reduction potentials can be compared for various respiratory reactions. This is useful because we can calculate a G for the reaction. See Section 8.3 Electron Tower and Energy H 2 + ½ O 2 => H 2 O The Electron Tower: Reduction potentials of half reactions H 2 2H+ + 2e- (Oxidation) (Reduction) 2H+ + 2e- + ½ O 2 H2O (Overall Rnx) H 2 + ½ O 2 => H 2 O E o = e- Electrons flow from low (more neg.) to high (more positive) potential e- donors. The greater the fall of electrons the more potential energy can be harvested in the balanced reaction. 11

12 Hydrogen Oxidation coupled to Oxygen reduction Example H 2 2H+ + 2e- (Oxidation) (Reduction) 2H+ + 2e- + ½ O 2 H2O (Overall Rnx) H 2 + ½ O 2 => H 2 O E o = E o (e- acceptor) - E o (e- donor) Nernst Equation to calculate G o G o = -nf E o Free Energy and Reactions Free energy change (ΔG) is the amount of energy in a system that is available to do work A ΔG indicates that the reaction is favorable and will proceed spontaneously (i.e., the reaction is exergonic) A ΔG indicates that the reaction is unfavorable and will only proceed if energy is supplied (i.e., the reaction is endergonic) 12

13 Electron transport chains and their relation to E 0 '. low E o electron flow hi E o 2H+ H+ H+ Q 2NADH 2NAD+ 2H+ H+ O 2 4H+ H+ 2 H 2 O per cyto Transfer electrons from an electron donor to an acceptor with a greater, (more positive) reduction potential. Electrons from NADH and FADH2 are transported in a series of redox reactions to a terminal electron acceptor Conserve some of the energy released during electron transfer in PMF Use PMF to synthesize ATP Example: Aerobic electron transport chain NADH + H + NAD + E o = V 2e -, 2H + 1/2O 2 H 2 O E o = V How much energy is released? 13

14 NADH + H + NAD + E o = V 2e -, 2H + 1/2O 2 H 2 O E o = V How much energy does it take to make 1 ATP? If G o = -31 kj/mol for ATP >>> ADP + Pi, how many ATPs can be made from -220 kj/mol rxn with NADH oxidation coupled to oxygen reduction? Theoretical: Reality: Efficiency: 14

15 Anaerobic E o Electron Tower 0.45 V n= 2e V n= 2e- Aerobic 1.24 V n= 2e- calculate the E o and G o Electron transport and ATP synthesis 15

16 Inhibitors of Respiration : block the flow of through the system, which blocks formation of. Carbon monoxide and cyanide bind to cytochromes DCCP (dicyclohexylcarbodiimide) binds to ATP synthase : Prevent without affecting. Dinitrophenol (DNP), lipid soluble make membrane leaky; destroys the PMF; shuts down ATP production by oxidative phosphorylation. PMF can be used for lots of processes 16

17 Three Important Processes to Remember in Respiration 1. Carbon Flow As an organic compound is oxidized to CO2, reducing power (NADH, NAD(P)H, FADH) and carbon intermediates are generated. These intermediates will be used in biosynthesis and/or secreted. 2. Electron Flow Electrons in a chemical energy source are transferred by the membrane-bound intermediate electron carriers of an ETS to a final electron acceptor (e.g. O 2, NO 3-, SO 4 2-,CO 2, ); The electron flow generates PMF. The reduced products are secreted 3. Oxidative Phosphorylation Energy generated by electron flow is captured as PMF and, then, used to synthesize ATP. Carbon and Electron Flow explain how Glycolysis and the TCA Cycle are linked to Oxidative Phosphorylation in Respiration. Summary Catabolism-the breakdown of larger, more complex molecules into smaller, simpler ones, during which energy is released, trapped, and made available for work Catabolism is a Multi-stage process Stage 1-breakdown of large molecules (polysaccharides, lipids, proteins) into their component constituents with the release of little (if any) energy 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 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 Substrate level Phosphorylation transfer of Pi from a high energy phosphorylated intermediate to ADP by a kinase enzyme fermentations are important pathways for SLP reactions. Respiration, Electron Transport, and Oxidative Phosphorylation Electrons from NADH and FADH2 are transported in a series of redox reactions to a terminal electron acceptor Electron carriers are located within the plasma membrane in prokaryotes Some of the energy liberated during electron transport is used to drive the synthesis of ATP in a process called oxidative phosphorylation Redox reactions Oxidation-reduction (redox) reactions involve the transfer of electrons from a donor (reducing agent or reductant) to an acceptor (oxidizing agent or oxidant) 17