Microbiology IIMicrobial physiology II –Some principles and mechanisms in the the central carbon metabolism
Christopher Bräsen
Lecture Plan
17.10. 2017 Mikrobielle Physiologie I ‐ Energetik Bräsen
24.10. 2017 Mikrobielle Physiologie II – Einige Prinzipien und Mechanismen im zentralen Kohlenstoffmetabolismus
Bräsen
31.10. 2017 Keine Vorlesung Bräsen
07.11. 2017 Mikrobielle Physiologie III – Nitrat‐Atmung Bräsen
14.11. 2017 Mikrobielle Physiologie IV – Acetogenese und der Acetyl‐CoA/Kohlenmonoxid Dehydrogenase‐Weg
Bräsen
21.11. 2017 Mikrobielle Physiologie V – Anaerobe Nahrungskette und Methanogenese Bräsen
28.11. 2017 Mikrobielle Physiologie VI – Sulfate Reduktion Bräsen
05.12. 2017 Antibiotika (Penicillium notatum) Meckenstock
12.12. 2017 Mikroorganismen in der Umwelt (Geobacter metallireducens) Meckenstock
19.12. 2017 Mikrobielles Wachstum (Elusimicrobium minutum) Meckenstock
09.01. 2018 Mikrobielle Fortbewegung (Thioploca) Meckenstock
16.01. 2018 Viren (T4) Meckenstock
23.01. 2018 Geschichte der Mikrobiologie Meckenstock
30.01. 2018 Wrap up/Ausweichtermin Meckenstock/Bräsen
Questions 1
• 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?• Which are the two basal mechanisms of ATP synthesis and what are their
characteristics? • 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.• How can ΔG values be calculated from Redoxpotentials, give the
equation?• Name the sites of SSP in glycolysis? Which enzyme catalyses the only
oxidation reaction in this pathway?• What is the frequently used cosubstrate in dehydrogenase catalyzed
reactions?
Example: Aerobic Glucose oxidation
Glucose = S1red. 6 O2 = S2ox.
6 CO2 = P1ox. 12 H2O = P2red.
ATP ATP
12 x 2[H]
Oxidative part Reductive part
ATP synthesis viaSubstrate levelphosphorylation(SSP)
ATP synthesis viaElectron transportphosphorylation(ETP)
Glucose (red.) + 6 O2 (ox.) → 6 CO2 (ox.) + 6H2O (red.)
Most energy yielding catabolic rections are redox reactions
Electron donating half reaction Electron accepting half reaction
Example: Aerobic Glucose oxidation
Glucose = S1red. 6 O2 = S2ox.
6 CO2 = P1ox. 12 H2O = P2red.
ATP ATP
12 x 2[H]
Oxidative part Reductive part
ATP synthesis viaSubstrate levelphosphorylation(SSP)
ATP synthesis viaElectron transportphosphorylation(ETP)
Glucose (red.) + 6 O2 (ox.) + 6 H2O → 6 CO2 (ox.) + 12 H2O (red.)
Most energy yielding catabolic rections are redox reactions
Electron donating half reaction Electron accepting half reaction
6 H2O
Aerobic glucose degradation
Glucose
6 O2
4 CO2
12 H2O
~34 ATP(ETP)
24 [H]
Oxidative part
Reductive part
Transport
Embden‐Meyerhof pathwayEntner‐Doudoroff pathway
Pyruvatedehydrogenasecomplex
Citric acid cycle
Glucose
2 Acteyl‐CoA
2 Pyruvate
4 [H]
4 [H]
16 [H]
2 CO2
1‐2 ATP(SSP)
2 ATP(SSP)
10 NADH → 100 H+
2 UQH2 → 12 H+
Respiratory chain
Σ 4 ATP (SLP) + 10 NADH + 2 UQH2
Σ ~38 ATP
Transport
ΔG = ΔµH+ = F ΔΨ – 2.3 RT ΔpH (i‐o)
ΔG = ΔµH+ = ~18 kJ/mol→ provides the energy to transport a substrate against a gradientof 1000; 50 kJ/mol = 1 ATP → concentration gradient 108
ΔG FΔΨ 2.3RTlgH out
H in
In case of a non electrogenic transport: ΔG 2.3RTlgS out
S in5.7
kJmol lg
S out
S in
A concentration gradient of 0.1 (10fold higher intracellular conc. of e.g. a growthsubstrate)
→ 5.7 kJ/mol has to be invested to transportthe substrate against this gradient of 10fold
ΔG 5.7kJmol lg
110 = ‐5.7 kJ/mol x ‐1
100fold → 11.4 kJ/mol1000fold → 17.1 kJ/mol
Sugar transport in Bacteria
H+out
Glucoseout
H+in
Glucosein
Glucoseout Glucosein
Maltoseout MaltoseinATP
ADP + Pi
K
K
F
GE
BA
HPr E1
CGlucoseout Glucose‐6‐P
PEP (=1 ATP)
Pyruvate
Membraneout + in ‐
Glucose‐6‐P
Glucose‐6‐P
= 1/3 ATP
ATP ADP
ATP ADP
Facilitated Diffusion
H+ Symport
PTS System
~P
ABC Transport
Rare in bacteria, e.g. glucose uptakeZymomonas mobilis, glycerol E. coli
Many aerobic bacteria
Many anaerobic, facultative bacteria
Widespread in all domains of life for a variety of substrates;
e.g. Maltose transporter in E. coli
Aerobic glucose degradation
Glucose
6 O2
4 CO2
12 H2O
~34 ATP(ETP)
24 [H]
Oxidative part
Reductive part
Transport
Embden‐Meyerhof pathwayEntner‐Doudoroff pathway
Pyruvatedehydrogenasecomplex
Citric acid cycle
Glucose
2 Acteyl‐CoA
2 Pyruvate
4 [H]
4 [H]
16 [H]
2 CO2
1‐2 ATP(SSP)
2 ATP(SSP)
10 NADH → 100 H+
2 UQH2 → 12 H+
Respiratory chain
Σ 4 ATP (SLP) + 10 NADH + 2 UQH2
Σ ~38 ATP
Glycolysis(Embden‐Meyerhof‐Parnas (EMP) patwhay)
Glycolysis (EMP patwhay)
Glycolysis (EMP patwhay)
Glycolysis (EMP patwhay)
Bar‐Even et al., 2012
Constraints:• preventing loss of carbon and energy→
Phosphorylation reduces permeability• C‐C bond cleavage is easier in β position
to carbonyl group• Avoiding toxic intermediates
Aims:• building blocks• Max. ATP gain (as much as possible)
Glycolysis (EMP patwhay)
1. Permeability
2. Aldol cleavage, C‐C bond cleavage iseasier in β position to carbonyl group
3. Permeability, both final C3 cleavageproducts are phosphorylated
5. building blocks, DHAP is a precursore.g. for lipid synthesis
4. Aldol cleavage, finally yields twoidentical C3 sugar phosphates (GAP)
Glycolysis (EMP patwhay)
6. Phosphate anhydride formation, NAD avoids toxic compound formation
7. Substrate level phosphorylation; 3PG isbuilding block in AA synthesis (Ser, Gly, Cys)
8.+9. Enables phosphate activation, andSLP in 10.; PEP is building block in AA synthesis (Tyr, Trp)
10. Substrate level phosphorylation; pyruvate is building block in AA synthesis(Ala, Leu, Ile, Lys, Val)
Glycolysis (EMP patwhay)
A second glycolytic route: The Entner‐Doudoroff (ED) pathway
• 1 ATP spent for Glucose phosphorylation• 2 ATP gained from the oxidation from one
molecule of GAP to pyruvate• Net yield 1 ATP and 2 NAD(P)H + H+
• 2‐keto‐3‐deoxy‐6‐phosphogluconate (KDPG) isthe key intermediate
• GAP is further oxidized by the same reactionsas in the EMP
The Entner‐Doudoroff (ED) pathway
The Entner‐Doudoroff (ED) pathway
• 1 ATP spent for Glucose phosphorylation• 2 ATP gained from the oxidation from one
molecule of GAP to pyruvate• Net yield 1 ATP and 2 NAD(P)H + H+
Avi Flamholz et al. PNAS 2013;110:10039-10044
The Entner‐Doudoroff (ED) pathway
Avi Flamholz et al. PNAS 2013;110:10039-10044
The Entner‐Doudoroff (ED) pathway
Avi Flamholz et al. PNAS 2013;110:10039-10044
The Entner‐Doudoroff (ED) pathway
Avi Flamholz et al. PNAS 2013;110:10039-10044
The Entner‐Doudoroff (ED) pathway
Avi Flamholz et al. PNAS 2013;110:10039-10044
• ED utilizers are in most cases aerobes (this does not mean that all aerobes areED utilizers)
• Some facultative anaerobes (e.g. E. coli) utilize this pathway for gluconatedegradation
• Widespread in strict aerobes like Pseudomonads
• Sugar transport as H+ symport• Exception Zymomonas mobilis
The Entner‐Doudoroff (ED) pathway
• 1 ATP spent for Glucose phosphorylation
• 2 ATP gained from the oxidation fromone molecule of GAP to pyruvate
• Net yield 1 ATP and 2 NAD(P)H + H+
• Widespread in strict aerobes like Pseudomonads
• Sugar transport as H+ symport• Exception Zymomonas mobilis
Aerobic glucose degradation
Glucose
6 O2
4 CO2
12 H2O
~34 ATP(ETP)
24 [H]
Oxidative part
Reductive part
Transport
Embden‐Meyerhof pathwayEntner‐Doudoroff pathway
Pyruvatedehydrogenasecomplex
Citric acid cycle
Glucose
2 Acteyl‐CoA
2 Pyruvate
4 [H]
4 [H]
16 [H]
2 CO2
1‐2 ATP(SSP)
2 ATP(SSP)
10 NADH → 100 H+
2 UQH2 → 12 H+
Respiratory chain
Σ 4 ATP (SLP) + 10 NADH + 2 UQH2
Σ ~38 ATP
Pyruvate dehydrogenase complex
• 3 Enzymes • 5 Coenzymes• Irreversible!
Aerobic glucose degradation
Glucose
6 O2
4 CO2
12 H2O
~34 ATP(ETP)
24 [H]
Oxidative part
Reductive part
Transport
Embden‐Meyerhof pathwayEntner‐Doudoroff pathway
Pyruvatedehydrogenasecomplex
Citric acid cycle
Glucose
2 Acteyl‐CoA
2 Pyruvate
4 [H]
4 [H]
16 [H]
2 CO2
1‐2 ATP(SSP)
2 ATP(SSP)
10 NADH → 100 H+
2 UQH2 → 12 H+
Respiratory chain
Σ 4 ATP (SLP) + 10 NADH + 2 UQH2
Σ ~38 ATP
The Citric acid cycle
• Acetyl‐CoA oxidation to CO2 andgeneration of reducing power → respiratory chain/energy via ETP (3 NADH + 1 UQH2 per acetyl‐CoA)
• 1 ATP/acetyl‐CoA via SSP:Enzyme + Succinyl‐CoA + Pi Enzyme(Succinyl~P) + HS‐CoA
Enzyme(Succinyl~P) Enzyme‐His246α~P + Succinate
Enzyme‐His246α~P + ADP E + ATP
• One C2 compound enters the cyleand two CO2 are formed→ the C2 compound is thus completelyoxidized
CH3CO‐S‐CoA + 3 H2O 2 CO2+ 8[H] + HS‐CoA
The Citric acid cycle
• The first step the aldolkondensationof acetyl‐CoA and oxaloacetate isirreversibel/highly exergonic ΔG0‘ = ‐32 kJ/mol
• Due to thioester hydrolysis• Not coupled to energy formation
CH3CO‐S‐CoA + 3 H2O + ADP + Pi 2 CO2+ ATP + 8[H] + HS‐CoA
The Citric acid cycle
‐190 mV
ΔG0‘ = ‐ n F ΔE0‘ΔE0‘ =(E0‘[Akzeptor]‐ E0‘[Donor])
ΔG0‘ = ‐ n F (‐0.320 V – (‐0,19 V))= ‐200 kJ/mol V * (‐0.13 V)= + 26 kJ/mol
• Reaction is endergonic• Equilibrium lies far on the side
of malate
At which ratio ofoxaloacetate/malate is the reductionof NAD+ feasable?
The Citric acid cycle
ΔG0‘ = ‐ n F ΔE0‘ΔE0‘ =(E0‘[Akzeptor]‐ E0‘[Donor])
ΔG0‘ = ‐ n F (‐0.320 V – (‐0,19 V))= ‐200 kJ/mol V * (‐0.13 V)= + 26 kJ/mol
• Reaction is endergonic• Equilibrium lies far on the side
of malate
At which ratio ofoxaloacetate/malate is the reductionof NAD+ feasable?
E‘ E0‘ ..
E‘ V E0‘ , lg ..
‐0,32 V 0,19 V , lg ..
-0,32 0,19 /0.03 lg ..
-0,32 0,19 /0.03 lg ..
-4.3 lg ..
~ 10-4 ..
The Citric acid cycle
• The first step the aldolkondensationof acetyl‐CoA and oxaloacetate isirreversibel/highly exergonic ΔG0‘ =‐32 kJ/mol
• Due to thioester hydrolysis• Not coupled to energy formation
CH3CO‐S‐CoA + 3 H2O + ADP + Pi 2 CO2+ ATP + 8[H] + HS‐CoA
→ the oxaloacetate concentration has tobe kept very low (~ four orders ofmagnitude below malate) to drivemalate oxidation with NAD+
→ accomplished by citrate synthasecatalzed exergonic reaction whichwithdraw oxaloacetate and pulls theequilibrium towards malate oxidation
The Citric acid cycle
• Rearragement from Citrate toisocitrate from a tertiary to a secondary alcohol
• With a tertiary alcohol hydrideabstraction is not possible becaus a free hydrogen atom at the hydroxyl C is missing
+ H‐ + H+
AldehydeAlcohol
Malate→ OxaloacetateIsocitrate→ Oxalosuccinate
The Citric acid cycle
CH3CO‐S‐CoA + 3 H2O + ADP + Pi 2 CO2+ ATP + 8[H] + HS‐CoA
‐500 mV
• α‐Ketoglutarate Dehydrogenase catalyses the second irreversible reaction of the cycle
• Similar (molecular and catalytic) topyruvate DH complex
• 1 ATP/acetyl‐CoA via SSP:Enzyme + Succinyl‐CoA + Pi Enzyme(Succinyl~P) + HS‐CoA
Enzyme(Succinyl~P) Enzyme‐His246α~P + Succinate
Enzyme‐His246α~P + ADP E + ATP
• Closed cycle only with a terminal electron acceptor more positive than+30 mV
+30 mV
Electron transferring coenzymes andprosthetic groups: Flavin nucleotides
• Flavoproteins• Isoalloxazin system• Can perform one and two electron transitions• Flavin is tightly bound in most flavoproteins = prosthetic group• Redox potential depends on the protein association: ‐400 mV – +100 mV• In succinate dehydrogenase near 0 mV (free FADH2 ‐220 mV)
The Citric acid cycle
CH3CO‐S‐CoA + 3 H2O + ADP + Pi 2 CO2+ ATP + 8[H] + HS‐CoA
‐500 mV
• α‐Ketoglutarate Dehydrogenase catalyses the second irreversible reaction of the cycle
• Similar (molecular and catalytic) topyruvate DH complex
• 1 ATP/acetyl‐CoA via SSP:Enzyme + Succinyl‐CoA + Pi Enzyme(Succinyl~P) + HS‐CoA
Enzyme(Succinyl~P) Enzyme‐His246α~P + Succinate
Enzyme‐His246α~P + ADP E + ATP
• Closed cycle only with a terminal electron acceptor more positive than+30 mV
• In succinate dehydrogenase FAD ~0 mV (FAD covalently bound), succinateDH transfers electrons from succinatefinally to the UQ pool in themembrane
+30 mV
The citric acid cycle as a metabolic hub
Anaplerotic reactions
PEP carboxykinase in Bacteriamore for gluconeogenesis
Aerobic glucose degradation
Glucose
6 O2
4 CO2
12 H2O
~34 ATP(ETP)
24 [H]
Oxidative part
Reductive part
Transport
Embden‐Meyerhof pathwayEntner‐Doudoroff pathway
Pyruvatedehydrogenasecomplex
Citric acid cycle
Glucose
2 Acteyl‐CoA
2 Pyruvate
4 [H]
4 [H]
16 [H]
2 CO2
1‐2 ATP(SSP)
2 ATP(SSP)
10 NADH → 100 H+
2 UQH2 → 12 H+
Respiratory chain
Σ 4 ATP (SLP) + 10 NADH + 2 UQH2
Σ ~38 ATP
Basic principle
Fig. 9.5 Biology (6th edition, Campbell & Reece)
Beispiel Zellatmung
Respiratory chain
Fig. 5.19 Brock Biology of Microorganisms (10th edition) (Madigan et al.)
Electron transfer coenzymes
Fig. 14.22 Essential Cell Biology (2nd edition, Alberts, Bray et al.)Fig. 5.12 Cell & Molecular Biology (4th edition, Karp)
Flavin-Mononukleotid Chinon Cytochrom Eisen-Schwefel-Zentren
Je 2 H+ und e- nur e-
Electron transport chain
• Electron transport chainmitochondria vs. E. coli
0.369 V → ~70 kJ/mol
0.209 V → ~40kJ/mol
0.563 V → ~100 kJ/mol
2 e‐
Komplex I
Komplex III
Komplex IV
Electron transport chain
http://watcut.uwaterloo.ca/webnotes/Metabolism/RespiratoryChain.html
Electron transport chain
Σ 10 H+/NADH→ ~3ATPΣ 6 H+/UQH2→ ~2 ATP
Mitochondria, Paracoccus denitrificans
Fumarate/Succinate +0,03 VFAD/FADH2 ~0.0 V
Komplex I NADH/ubiquinone oxidoreductase
Komplex III Ubichinone/Cytochrome c oxidoreductase (bc1 Komplex)
Komplex IV Cytochrome c oxidase
Komplex II Succinate dehydrogenase
Electron transport chainmitochondria vs. E. coli
2
2
Σ 10 H+/NADH→ ~3ATPΣ 6 H+/UQH2→ ~2 ATP
Σ 8 H+/NADH→ ~2 ATPΣ 4 H+/UQH2→ ~1 ATP
Mitochondria, Paracoccus denitrificans
E. coli
• without cyt c and bc1‐complex• No cyt c oxidase• Chinol oxidase
Branched respiratory chains
Cyt0 low oxygen affinityCytd high oxygen affinity
→ Fine tuning of respiratory chain in responce tooxygen concentrations
Electron transport chain
Σ 10H+/2e‐→ ~3ATP
ATP Synthase
Reversibel (ATPase)F1: 5 UE (α3β3γεδ)F0: 3 UE (ab2c12)Katalytische Aktivität: β UERotor: c12,γ, εStator: a, b2, δ
3/4 H+ transportiert pro ATP
Mechanism of ATP synthesis
Paul Boyer-Rotation der γ-UE zu α,β-Ring-L: loose site (ADP + Pi Bindung)-T: tight site (ATP Bildung)-O: open site (leer)
O
T
L
Question 2
• What is the frequently used cosubstrate in dehydrogenase catalyzed reactions? How areelectrons transfered?
• What are the four main steps in the oxidative part of aerobic glucose degradation? What isthe net gain of reduction equivalents and ATP?
• What are the main functions of glycolysis (EM pathway)? What is the substrate what theproduct? What happens in the preparatory phase? Name the sites of SSP in glycolysis (incl. Enzymes)? Which enzyme catalyses the only oxidation reaction in this pathway? What are theirreversible steps (incl. Enzymes)? Give the net yield of ATP and reduction equivalents.
• Name the differences between EM and ED pathway.
• Which enzymes and coenzymes take part in the pyruvate oxidation? Give the name of thecomplex.
• What is the basic function of the citric acid cycle? Which compound fit into the cycle andwhat are the products? What are the irreversible steps. Name the site of SSP in the cycle. Where is CO2 generated? What are the oxidation steps? Can electrons be transfered fromSuccinate to NAD+?
• How does ETP basically function? What is the difference in the electron transport chain ofmitochondria/Paracoccus and E. coli? What does that mean for the ATP yield?
Question 2
• Was ist das in Degydrogenase caztalysierten Reaktionen häufig verwendete Cosubstrat und wievieleElektronen überträgt es?
• In welche vier Hauptschritte kann man den aeroben Glucose‐Abbau einteilen? Was sind jeweils die (Netto)Ausbeuten an ATP und Reduktionsäquivalenten?
• Was sind die Hauptfunktionen der Glycolyse (EMP‐Weges)? Was ist das Substrat was sind die Produkte, geben Sie die Nettoausbeute an ATP und Reduktionsäquivalenten an? Was passiert in der Vorbereitungsphase? Welche Reaktionen der Glycolyse sind mit Substratstufenphosphorylierung verbunden, wie heißen die katalysierenden Enzyme? Welches Enzym katalysiert die einzige Oxidationsreaktion im EMP‐Weg? Welches sind die irreversiblen Schritte der Gylcolyse, welche Enzyme katalysieren sie?
• Benennen Sie die Unterschiede zwischen EMP‐ und ED‐Weg.
• Welche Enzyme und Coenzyme sind an der Pyruvat‐Oxidation beteiligt? Wie heißt der Enzym‐Komplex?
• Was sind die Hauptfunktionen des Citrat‐Zyklus? Welche Verbindung wird in den Zyklus eingeschleust und was sind die Produkte? Benennen Sie die irreversiblen Schritte. In welcher Reaktion wird ATP/GTP über SSP gebildet. Welches sind die CO2 generierenden Schritte? Welches sind die Oxidationsschritte? Können Elektronen von Succinat auf NAD+ übertragen werden?
• Wie funktioniert grundsätzlich die Elektronentransportphosphorylierung? Was ist der Unterschied zwischen den Elektronentransportketten der Mitochondrien bzw. von Paracoccus denitrificans und E. coli und was bedeutet das für die ATP‐Ausbeute?
Nitrate respiration („Homework“)
aGlucose + bNO3‐ + → cCO2 + dNO2
‐ +
Many organisms like e.g. E. coli and Paracoccus denitrificans grow with glucoseand NO3
‐ as electron donor and acceptor, respectively:
• How does the complete redox equation/balance look like?• How much energy is gained and how much ATP can maximally be synthetized?• Does complete glucose oxidation include a closed/complete TCA cycle?