Microbiology II Microbial physiology I Energetics

Size: px

Start display at page:

Download "Microbiology II Microbial physiology I Energetics"

Cornelia Davidson
6 years ago
Views:

1 Microbiology II Microbial physiology I Energetics Catabolism Heat Efficiency ~ 60% Efficiency ~ 40% Anabolism Chemical energy (chemotrophic) Light energy (phototrophic) ATP +/ 50 kj/mol ADP + P i Biosyntheses Transport Movement ΔG<0 exergonic Isothermal, irreversible conditions Heat Work ΔG>0 endergonic Christopher Bräsen

2 Lecture Plan Mikrobielle Physiologie I Energetik Bräsen Mikrobielle Physiologie II Einige Prinzipien und Mechanismen im zentralen Kohlenstoffmetabolismus Bräsen Keine Vorlesung Bräsen Mikrobielle Physiologie III Nitrat Atmung Bräsen Mikrobielle Physiologie IV Acetogenese und der Acetyl CoA/Kohlenmonoxid Dehydrogenase Weg Bräsen Mikrobielle Physiologie V Anaerobe Nahrungskette und Methanogenese Bräsen Mikrobielle Physiologie VI Sulfate Reduktion Bräsen Antibiotika (Penicillium notatum) Meckenstock Mikroorganismen in der Umwelt (Geobacter metallireducens) Meckenstock Mikrobielles Wachstum (Elusimicrobium minutum) Meckenstock Mikrobielle Fortbewegung (Thioploca) Meckenstock Viren (T4) Meckenstock Geschichte der Mikrobiologie Meckenstock Wrap up/ausweichtermin Meckenstock/Bräsen

3 Textbooks Fuchs Allgemeine Mikrobiologie (Thieme) Brock Mikrobiologie (Pearson) Campbell/Reece Biologie (Pearson) Stryer Biochemie (Spektrum) Nelson/Cox Lehninger: Principles in Biochemistry (W.H. Freeman and Company)

4 Questions 1 Which are the two basal mechanisms of ATP synthesis and what are their characteristics? In which two parts can the energy metabolism be devided? How much energy is required to synthesize ATP from ADP and Pi considering cellular concentrations of reactants? How much energy is approximately gained from one H + flowing back into the cell (e.g. E. coli)? How can ATP yields be estimated if the energy yielding catabolic reaction is known (overall efficiency of energy metabolism ~60%)? What are energy rich intermediates? Give three examples. Name the sites of SSP in glycolysis? Whichenzymecatalysestheonly oxidation reaction in this pathway? How can ΔG values be calculated from Redoxpotentials, give the equation? What is the frequently used cosubstrate in dehydrogenase catalyzed reactions?

5 Energy transformation Catabolism Heat Efficiency ~ 60% Efficiency ~ 40% Anabolism Chemical energy (chemotrophic) Light energy (phototrophic) ATP +/ 50 kj/mol ADP + P i Biosyntheses Transport Movement ΔG<0 exergonic Isothermal, irreversible conditions Heat Work ΔG>0 endergonic Catabolism and Anabolism are coupled through the adenylate system

6 Energetics as 1 + bs 2 cp 1 + dp 2 ΔG? ΔG 0 = [cδg f0 (P1) + dδg f0 (P2)] [aδg f0 (S1) + bδg f0 (S2)] In biological systems with protons as reactants: ΔG 0 (ph 7 ([10 7 M] instead of ph 0 [10 0 =1 M]) ΔG f 0 (H + ) = 0 ΔG f 0 (H + ) = 40 kj/mol ΔG = ΔG 0 + RT x ln [P 1] c [P 2 ] d [S 1 ] a [S 2 ] b ΔG = ΔG 0 + 5,7 kj/mol x lg [P 1 ] c [P 2 ] d [S 1 ] a [S 2 ] b

7 Example: Aerobic Glucose oxidation ΔG f0 [kj/mol) C 6 H 12 O 6 + O 2 CO 2

8 Example: Aerobic Glucose oxidation ΔG f0 [kj/mol) C 6 H 12 O 6 + O 2 6 CO 2

9 Example: Aerobic Glucose oxidation ΔG f0 [kj/mol) C 6 H 12 O O 2 6 CO H 2 O

10 Gibbs free energies of formation In Brock Mikrobiologie, 13. Auflage, Anhang

11 Gibbs free energies of formation In Brock Mikrobiologie, 13. Auflage, Anhang

12 Energetics

13 Example: Aerobic Glucose oxidation ΔG f0 [kj/mol) C 6 H 12 O O 2 6 CO H 2 O ΔG 0 = [6 ( 394) CO ( 237) H2O] [( 917) glucose + 6 (0) O 2 ] = [ ] = 2869 kj/mol How much ATP can be synthesized from this catabolic reaction?

14 ATP Why energy rich (=high ΔG of hydrolysis, high group transfer potential)? ATP Resonance stabilization of products of hydrolysis exceeds resonance stabilization of the compound itself Electrostatic repulsion between negatively charged phosphate oxygen atoms favors separation of the phosphates Stabilization through hydratation: Water can bind more efficiently to ADP and Pi than to the Anhydride bond in ATP

15 Energetics of ATP synthetsis How much energy is required for ATP synthesis? ADP 3 + P i 2 ATP 4 + H 2 O Standard conditions: ΔG 0 = +32 kj/mol Cellular conditions: ~10 mm ATP, ~1 mm ADP, ~10 mm P i ΔG = ΔG 0 + RT x ln [ATP]/[ADP][Pi] ΔG = +32 kj/mol kj/mol x lg 10 3 = +49 kj/mol

16 Constants and Units

17 Energy transformation Catabolism Heat Efficiency ~ 60% Efficiency ~ 40% Anabolism Chemical energy (chemotrophic) Light energy (phototrophic) ATP +/ 50 kj/mol ADP + P i Biosyntheses Transport Movement ΔG<0 exergonic Isothermal, irreversible conditions Heat Work ΔG>0 endergonic Catabolism and Anabolism are coupled through the adenylate system

18 Energetics of ATP synthetsis How much energy is required for ATP synthesis? ADP 3 + P i 2 ATP 4 + H 2 O Standard conditions: ΔG 0 = +32 kj/mol Cellular conditions: ~10 mm ATP, ~1 mm ADP, ~10 mm P i ΔG = ΔG 0 + RT x ln [ATP]/[ADP][Pi] ΔG = +32 kj/mol kj/mol x lg 10 3 = +49 kj/mol Regarding the irreversibility of the whole metabolism: Efficiency ~60% ~ kj/mol

19 Example: Aerobic Glucose oxidation ΔG f0 [kj/mol) C 6 H 12 O O 2 6 CO H 2 O ΔG 0 = [6 ( 394) CO ( 237) H2O] [( 917) glucose + 6 (0) O 2 ] = [ ] = 2869 kj/mol How much ATP can be synthesized from this catabolic reaction? 2869/ ATP

20 Mechanisms of ATP synthesis Most energy yielding catabolic rections are redox reactions as 1 (red.) + bs 2 (ox.) cp 1 (ox.) + dp 2 (red.) Oxidative part Reductive part Substrat 1 red. Substrat 2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) Produkt 1 ox. Produkt 2 red. (given: thermodynamic and mechanistic feasability (?))

21 Example: Aerobic Glucose oxidation Most energy yielding catabolic rections are redox reactions Glucose (red.) + 6 O 2 (ox.) 6 CO 2 (ox.) + 6H 2 O (red.) Oxidative part Reductive part Electron donating half reaction Electron accepting half reaction Glucose = S1 red. 6 O 2 = S2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) 6 CO 2 = P1 ox. 12 H 2 O = P2 red.

22 Energy metabolism Oxidative part Reductive part Substrat 1 red. Substrat 2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) Produkt 1 ox. Produkt 2 red. Cytoplasm (dehydrogenases, kinases) energy rich phosphoryl compounds natp/s1 Energy quantum 1 ATP = 50 kj/mol Membrane bound (ET proteins, ATP synthase) Electrochemical proton potential (ΔµH + =nh + /S2) Energy quantum 1H + =18 kj/mol (=1/3 ATP)

23 Substrate level phosphorylation The energy of a highly exergonic reaction (often oxidation of carbonyl to carboxyl groups) is used to form energy rich intermediates (phosphorylated intermediates), e.g. GAP 1,3BPG 3 PG, pyruvate (acetyl CoA acetyl~p) ( acetate)

24 Substrate level phosphorylation The energy of a highly exergonic reaction is used to form energy rich intermediates often through oxidation of carbonyl to carboxyl groups, e.g. GAP 1,3BPG 3 PG, pyruvate acetyl CoA acetyl~p acetate Energy rich intermediates are: Phosphate anhydrides (1,3 Bisphosphoglycerate, Acetyl P) Enolester (PEP) Thioesters Transfer phosphorylgroups to ADP

25 Substrate level phosphorylation The energy of a highly exergonic reaction is used to form energy rich intermediates often through oxidation of carbonyl to carboxyl groups, e.g. GAP 1,3BPG pyruvate acetyl CoA) Energy rich intermediates are: Phosphate anhydrides (1,3 Bisphosphoglycerate, Acetyl P) Enolester (PEP) Thioesters Transfer phosphorylgroups to ADP

26 Glyceraldehyde 3 phosphate oxidation ΔG ~ 50 kj/mol Glyceraldehyde 3 phosphate 3 Phosphoglycerate

27 Glyceraldehyde 3 phosphate Dehydrogenase

28 1,3 bisphosphoglycerate as energy rich intermediate 1,3 Bisphosphoglycerate = Carboxyl/Phosphoanhydride bond!

29 Substrate level phosphorylation Why energy rich (=high ΔG of hydrolysis, high group transfer potential)? Phosphoenolpyruvate (PEP), involved in ATP synthesis in glycolysis, has a very strongly negative ΔG of Pi hydrolysis (~ 52 kj/mol). Removal of Pi from ester linkage in PEP is spontaneous because the enol spontaneously converts to a ketone (Keto enol tautomerie, ~ 30 kj/mol).

30 Pyruvate Kinase

31 Pyruvate Kinase

32 Substrate level phosphorylation Why energy rich (=high ΔG of hydrolysis, high group transfer potential)? Thioester (e.g. Acetyl CoA) Resonance stabilization of products of hydrolysis exceeds resonance stabilization of the compound itself Stabilization through hydratation The thioester is more energy rich compared to the oxygen ester because the higher resonance stabilization of the latter

33 Substrate level phosphorylation Why energy rich (=high ΔG of hydrolysis, high group transfer potential)? Thioester (e.g. Acetyl CoA) Resonance stabilization of products of hydrolysis exceeds resonance stabilization of the compound itself Stabilization through hydratation The thioester is more energy rich compared to the oxygen ester because the higher resonance stabilization of the latter P i ADP acetyl CoA acetyl~p acetate HS CoA ATP

34 Energy metabolism Oxidative part Reductive part Substrat 1 red. Substrat 2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) Produkt 1 ox. Produkt 2 red. Cytoplasm (dehydrogenases, kinases) energy rich phosphoryl compounds natp/s1 Energy quantum 1 ATP = 50 kj/mol Membrane bound (ET proteins, ATP synthase) Electrochemical proton potential (ΔµH + =nh + /S2) Energy quantum 1H + =18 kj/mol (=1/3 ATP)

35 NAD + /NADH + H + Hydride ion transfer! NADH formation can be followed by observing the appearance of the absorbance at 340 nm (molar extinction coefficient 340 = 6,200 M 1 cm 1 ).

H + C OH GAP 3 Phosphglycerate Aldehyde H Aldehydehydrate Carboxylic acid + H + H + Malate Oxaloacetate

36 NAD(P) + /NAD(P)H + H + Dehydrogenase catalysed reactions: + Hydride anion ion transfer! Water soluble electron carrier Freely diffusable NADP in anabolism NAD in catabolism R O H +H 2 O + H + H + C OH GAP 3 Phosphglycerate Aldehyde H Aldehydehydrate Carboxylic acid + H + H + Malate Oxaloacetate Isocitrate Oxalosuccinate Alcohol Aldehyde FAD (Quinone) + H + H + Succinate Fumarate Single C C bond Double C=C bond

37 Energy metabolism Oxidative part Reductive part Substrat 1 red. Substrat 2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) Produkt 1 ox. Produkt 2 red. Cytoplasm (dehydrogenases, kinases) energy rich phosphoryl compounds natp/s1 Energy quantum 1 ATP = 50 kj/mol Membrane bound (ET proteins, ATP synthase) Electrochemical proton potential (ΔµH + =nh + /S2) Energy quantum 1H + =18 kj/mol (=1/3 ATP)

38 Electron transport phosphorylation In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (ΔpH ) and an electrical gradient (Δψ). The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton specific channels (F o ). The proton motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F 1 complex associated with F o. Electron transport chain Σ 10H + /2e ~3ATP

39 Electron transport phosphorylation At the cytoplasmic membrane impermeable to protons Coupled to the formation of an electrochemical proton potential = energy rich intermediate Electron transport chain Σ 10H + /2e ~3ATP

40 Electron transport phosphorylation Electrochemical proton potential ΔµH + gradient [H + ] o > [H + ] i ΔpH, acidic outside alkaline inside ΔΨ, electrogenic 3 4 H + /ATP ΔG = +50 kj ΔG= n F ΔE 50 kj = 0.13 V (0.17 V) 4 x 96.5 ( 3) ΔG = ΔµH + = ~17 kj/mol Brock Mikrobiologie Pearson 2013

41 Electrochemical proton potential ΔµH + gradient [H + ] o > [H + ] i ΔpH, acidic outside alkaline inside ΔΨ, electrogenic ΔG = ΔµH + = F ΔΨ RT ΔpH (i o) = 96.5 x 0.15V x 0.5 = 17.4 kj/mol E. coli: ΔpH ~ 0.5 ΔΨ ~ 0.15V ΔG/F = ΔµH + /F = ΔΨ RT/F ΔpH (i o) = Δp [V] proton motive force Δp = ΔΨ + Z ΔpH (i o) Z = 2.3 RT/F = V The poten al difference of at least 0.13 V 0.17 V corresponds to a ph difference of ~2.2 3 Brock Mikrobiologie Pearson 2013

42 Electrochemical proton potential ΔµH + gradient [H + ] o > [H + ] i ΔpH, acidic outside alkaline inside ΔΨ, electrogenic ΔG = ΔµH + = F ΔΨ RT ΔpH (i o) E. coli: ΔpH ~ 0.5 ΔΨ ~ 0.15V ΔG = ΔµH + = 96.5 kj/mol V 0.15 V kj/mol 0.5 (i o) ΔG = ΔµH + = ~18 kj/mol Brock Mikrobiologie Pearson 2013

43 Electron transport chain Σ 10H + /2e ~3 ATP

44 Example: Aerobic Glucose oxidation Oxidative part Reductive part Glucose 6 O 2 ATP synthesis via Substrate level phosphorylation (SSP) ATP 12 x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) 6 CO 2 12 H 2 O Cytoplasm (dehydrogenases, kinases) energy rich phosphoryl compounds natp/s1 Energy quantum 1 ATP = 50 kj/mol Membrane bound (ET proteins, ATP synthase) Electrochemical proton potential (ΔµH + =nh + /S2) Energy quantum 1H + =18 kj/mol (=1/3 ATP)

45 Example: Aerobic Glucose oxidation Most energy yielding catabolic rections are redox reactions as 1 (red.) + bs 2 (ox.) cp 1 (ox.) + dp 2 (red.) Oxidative part Reductive part Electron donating half reaction Electron accepting half reaction Glucose = S1 red. 6 O 2 = S2 ox. ATP synthesis via Substrate level phosphorylation (SSP) ATP n x 2[H] ATP ATP synthesis via Electron transport phosphorylation (ETP) 6 CO 2 = P1 ox. 12 H 2 O = P2 red.

46 Redox potentials The redox potenial indicate the tendency of a redox couple to donate electrons to (=reduce) or to accept electrons from (=oxidize) the standard hydrogen electrode. E 0 (2 H + /H 2 ) = 0 mv (under Standard conditions 25 C, 1 M ) E 0 (X ox /X red ) < 0 tends to reduce E 0 (X ox /X red ) > 0 tends to oxidize (H + /H 2 ) E 0 (2 H + /H 2 ) = 420 mv (under Standard conditions 25 C, 1 M, ph 7) E E 0.. E V E 0, lg..

47 ΔG and redox potentials ΔG 0 = n F ΔE 0 ΔE 0 =(E 0 [Akzeptor] E 0 [Donor])

48 Example: Aerobic Glucose oxidation as 1 (red.) + bs 2 (ox.) cp 1 (ox.) + dp 2 (red.) Oxidative part Reductive part Electron donating half reaction Electron accepting half reaction Glucose = S1 red. 6 O 2 = S2 ox. ΔG 0 = n F ΔE 0 ΔE 0 =(E 0 [Akzeptor] E 0 [Donor]) ATP (SSP) n x 2[H] ATP (ETP) 6 CO 2 = P1 ox. 12 H 2 O = P2 red. 430 mv +820 mv ΔG 0 = 24 x 96.5 kj/mol V x ( ( 0.43)) = 2895 kj/mol

49 Example: Aerobic Glucose oxidation Oxidative part Reductive part How much energy is gained from oxidative and reductive part, respectively? Electron donating half reaction Glucose = S1 red. Electron accepting half reaction 6 O 2 = S2 ox. Can be estimated if the electron carrier (incl. E 0 ) is known. ATP n x 2[H] ATP Assumption: NAD + /NADH ( 0.32 V) is the sole electron carrier (as a proxy). 6 CO 2 = P1 ox. 12 H 2 O = P2 red. 430 mv +820 mv

50 Example: Aerobic Glucose oxidation First half reaction: 430 mv 320 mv Glucose (red.) + 12 NAD + (ox.) + 6 H 2 O 6 CO 2(ox.) + 12 NADH (red.) + 12 H + How energy is gained from oxidative and reductive part, respectively? Can be estimated if the electron carrier (incl. E 0 ) is known. Assumption: NAD + /NADH ( 0.32 V) is the sole electron carrier (as a proxy). ΔG 0 = n F ΔE 0 ΔE 0 =(E 0 [Akzeptor] E 0 [Donor]) E 0 (CO 2 /glucose) = 430 mv E 0 (NAD+/NADH) = 320 mv ΔG 0 = 24 x 96.5 kj/mol V x ( 0.32 V ( 0.43 V)) = 255 kj/mol 4 ATP via SSP

51 Example: Aerobic Glucose oxidation Second half reaction: 320 mv +820 mv 12 NADH (red.) + 12 H O 2 (ox.) 12 NAD + (ox.) + 12 H 2O (red.) How energy is gained from oxidative and reductive part, respectively? Can be estimated if the electron carrier (incl. E 0 ) is known. Assumption: NAD + /NADH ( 0.32 V) is the sole electron carrier (as a proxy). ΔG 0 = n F ΔE 0 ΔE 0 =(E 0 [Akzeptor] E 0 [Donor]) E 0 (O 2 /H 2 O) = +820mV E 0 (NAD+/NADH) = 320 mv ΔG 0 = 24 x 96.5 kj/mol V x (0.82 V ( 0.32 V)) = 2640 kj/mol 34 ATP via ETP

52 Aerobic glucose degradation Oxidative part Glucose Σ 4 ATP (SLP) + 10 NADH + 2 UQH 2 Transport Σ ~38 ATP Embden Meyerhof pathway Entner Doudoroff pathway Glucose 2 Pyruvate 4 [H] 1 2 ATP (SSP) Reductive part 6 O 2 Pyruvate dehydrogenase complex 2 Acteyl CoA 4 [H] 2 CO 2 24 [H] 10 NADH 100 H + 2 UQH 2 12 H + Respiratory chain ~34 ATP (ETP) Citric acid cycle 16 [H] 2 ATP (SSP) 12 H 2 O 4 CO 2

53 Questions 1 Which are the two basal mechanisms of ATP synthesis and what are their characteristics? In which two parts can the energy metabolism be devided? How much energy is required to synthesize ATP from ADP and Pi considering cellular concentrations of reactants? How much energy is approximately gained from one H + flowing back into the cell (e.g. E. coli)? How can ATP yields be estimated if the energy yielding catabolic reaction is known (overall efficiency of energy metabolism ~60%)? What are energy rich intermediates? Give three examples. Name the sites of SSP in glycolysis? Whichenzymecatalysestheonly oxidation reaction in this pathway? How can ΔG values be calculated from Redoxpotentials, give the equation? What is the frequently used cosubstrate in dehydrogenase catalyzed reactions?

54 Exercize: Anaerobic Glucose oxidation ( O 2, without exogenous electron acceptor) ΔG f0 [kj/mol) C 6 H 12 O 6 C 3 H 5 O 3 (Lactate) Give the complete reaction equation. Calculate the standard free energy (ph 7). How many ATP can maximally be synthesized? Compare and discuss in the light of the aerobic degradation.

Lecture Series 9 Cellular Pathways That Harvest Chemical Energy

Lecture Series 9 Cellular Pathways That Harvest Chemical Energy Reading Assignments Review Chapter 3 Energy, Catalysis, & Biosynthesis Read Chapter 13 How Cells obtain Energy from Food Read Chapter 14