Respiration

Question 1

Describe the mitochondrial electron transport chain

Answer 1

1. Complex I oxidises NADH, Complex II oxidises succinate, reducing quinone to quinol
2. Complex III is reduced via quinol oxidation
3. 2 molecules of complex III reduce complex IV
4. Complex IV reduces O2 to H2O, generating PMF
5. Complex V (ATP Synthase) uses PMF to generate ATP

Question 2

What are the key differences between METC and the ETC in bacteria.

Answer 2

Instead of complex I/II, bacteria use Nuo and Ndh-2 to oxidise NADH, Sdh to oxidise succinate.
Complex III/IV are replaced with a single complex of either Cytochrome bo3 or Cytochrome bd.
Since bacteria do not have mitochondria, the ETC occurs in the cytoplasm with PMF being generated across the inner membrane, high H+ concentration in the periplasm and low H+ concentration in the cytoplasm.

Question 3

Tell me about Nuo

Answer 3

Nuo is reduced by NADH, it translocates 4H+ across the membrane. It is made of 14 subunits, NuoA-N, ~550kDa, 64TMHs, 9FeS clusters, FMN cofactor is key to e- entry into the complex.

Question 4

Tell me about Ndh-2

Answer 4

Ndh-2 is reduced by NADH, it is not a proton pump. It is a single subunit, ~45kDa, has no TMHs as it’s a peripheral protein. It has 1FAD cofactor.

Question 5

Tell me about Sdh

Answer 5

Sdh is reduced by the conversion of Succinate to Fumarate, using FAD as an intermediate. It is not a proton pump. It is made up of 4 subunits, SdhA-D, ~150kDa, 6TMHs, 1FAD, 3FeS, 1Heme b

Question 6

Tell me about Cyo

Answer 6

Cytochrome bo3 is reduced by quinol, releasing 2H+ into the periplasm and translocating an additional 2H+ across the membrane. It is made up of 4 subunits, CyoA-D. It’s a haem-copper oxidase with haem b, haem o3 and a Cu centre. It has a lower affinity to oxygen, working under oxic conditions.

Question 7

Tell me about Cyd

Answer 7

Cytochrome bd is reduced by quinol, releasing 2H+ into the periplasm. It is not a proton pump. It is made up of 4 subunits, CydA,B,H,X, 2 heme b, 1 heme d. It has a high affinity to oxygen working under microoxic conditions. It is more resistant to sulphides, H2O2 and nitric oxides.

Question 8

Tell me about Nuo/Cyo as a combination

Answer 8

This combination produces the highest H+/e- ratio; 8H+ is translocated across the membrane per NADH oxidised (4H+/e-). E. coli ATP synthase uses 10H+ to synthesize 3ATP; 2.4ATP/NADH.

Question 9

Tell me about Ndh/Cyo as a combination

Answer 9

This combination translocates 4H+ into the periplasm per NADH oxidised (2H+/e-). E. coli ATP synthase uses 10H+ to synthesize 3ATP; 1.2ATP/NADH.

Question 10

When is Ndh/Cyo used over Nuo/Cyo

Answer 10

Ndh/Cyo can handle a higher metabolic flux. During periods of growth, E. coli will prioritise speed over efficiency as growth conditions may only be transient. However, under microaerobic conditions or under anaerobic conditions with alternate e- acceptors Nuo is used.

Question 11

Why are Ndh & Cyd antibiotic targets

Answer 11

They aren’t present in humans but are present in many pathogens such as Mycobacterium tuberculosis, Salmonella, Listeria, Shigella flexneri, Plasmodium falciparum, Toxoplasma gondii

Question 12

Give some examples of anaerobic e- donors

Answer 12

Formate to CO2
H2 to H+
NADH to NAD+
Lactate to Pyruvate
Gly-3-P to DHAP

Question 13

Which quinones are used in anaerobic respiration?

Answer 13

Napthaquinones: Menaquinone to Menaquinol (MK to MKH2)

Question 14

Give some examples of anaerobic e- acceptors

Answer 14

Succinate to Fumarate
TMA to TMAO
DMS to DMSO
NH4 to NO2- (Ammonia to nitrite)
NO2- to NO3- (nitrate)

Question 15

How is pyruvate processed under anaerobic conditions?

Answer 15

Under anaerobic conditions, pyruvate dehydrogenase is inhibited. Instead, pyruvate formate lyase (PFL, which is normally inhibited by O2) converts Pyruvate into Formate and AcetylCoA.

AcetylCoA is converted to Acetate producing 1 ATP via substrate-level phosphorylation, the Acetate is excreted

Formate is an e- donor at Fdn (Formate dehydrogenase)

Question 16

What are the oxidative and reductive branches of the TCA under anaerobic conditions?

Answer 16

Oxidative branch:
Citrate to Isocitrate to α-ketoglutarate, 1 NADH is produced

Reductive branch:
PEP + CO2 to Oxaloacetate to Malate to Fumarate to Succinate, 1 NADH and 1 FADH is used.

Question 17

Tell me about Fdn

Answer 17

Formate dehydrogenase is a multisubunit enzyme with the cofactors:
5FeS clusters, 2 Mo[MGD], 2 Heme b

It is reduced by the conversion of Formate to CO2 + 2H+ in the periplasm, it uses the 2e- to reduce menaquinone.

2H+ added to the periplasm.

Question 18

Tell me about Frd

Answer 18

Fumarate reductase is a multisubunit enzyme with the cofactors:
3FeS clusters, Flavin

It is reduced by menaquinol, releasing 2H+ back into the cytoplasm due it’s lack of redox loop.

No net H+ change

Question 19

Tell me about Nar

Answer 19

Nitrate reductase (NarGHI) is a multisubunit enzyme with the cofactors:
5FeS cluster, 2 Mo[MGD], 2 Heme b

It is reduced by menaquinol, releasing 2H+ into the periplasm. It reduces Nitrate in the cytoplasm, using 2H+ to form Nitrite.

2H+ moved to the periplasm.

Question 20

Tell me about Nap

Answer 20

Nitrate reductase (NapABC) is a multisubunit enzyme with the cofactors:
1FeS cluster, 2Mo[MGD], 6 c-type cytochromes

It is reduced by menaquinol, releasing 2H+ into the periplasm. It reduces Nitrate in the periplasm, using 2H+ to form Nitrite.

No net change in H+.

Question 21

Tell me about Nuo/Frd as a combination

Answer 21

This combination translocates 4H+/2e- generating 1.2ATP/NADH.

All of the changes in H+ occur at Nuo with Frd not contributing to PMF but instead ensuring redox balance.

Question 22

Tell me about Fdn/Nar as a combination

Answer 22

This combination translocates 4H+/2e- generating 1.2ATP/Formate.

Fdn and Nar both add 2H+ to the periplasm each.

Question 23

Tell me about Fdn/Nap as a combination

Answer 23

This combination translocates 2H+/2e- generating 0.6ATP/Formate.

Fdn adds 2H+ to the periplasm while Nap ensures redox balance.

Question 24

Tell me about respiration in Paracoccus denitrificans

Answer 24

It is a metabolically versatile G- soil bacterium with an ETC very similar to the METC.
Has 2 additional terminal oxidases which allows it to use C1 compounds as e- donors and H2O2 as an e- acceptor.

Question 25

Tell me about respiration in Helicobacter pylori

Answer 25

G- microaerophilic helical bacteria in the stomach (exclusively).
It has just 1 high affinity oxidase.
Fumarate and H2O2 are alternate e- acceptors.
Cytochrome bc1 oxidases MKH2.
It doesn’t have an SDH.
Frd may be able to act in reverse.
Flavodoxin acts as an e- donor via complex I.

Question 26

Tell me about respiration in Campylobacter jejeuni

Answer 26

G- microaerophilic helical & highly motile bacteria which cause gastroenteritis.
They have a highly branched respiratory ETC with a wide range of e- donors and e- acceptors in addition to O2.

Question 27

Tell about fermentation in bacteria

Answer 27

In the absence of O2 and a respiratory ETC, endogenous e- acceptors are used. ATP production is limited to substrate-level phosphorylation in the cytoplasm. Pyruvate, or a derivative acts as an organic e- acceptor, therefore NADH must be generated to maintain glycolysis. Most carbon substrate is routed towards fermentation products which are excreted as waste, 5x less biomass is generated with a low ATP yield of 1-3ATP/Glucose.

Question 28

Tell me about fermentation in muscle tissue

Answer 28

In the absence of O2, pyruvate remains in the cytoplasm and is reduced to lactate. NADH is the e- donor. This yields 2ATP/Glucose

Question 29

Tell me about homolactic fermentation

Answer 29

It occurs in Gram positive bacteria such as lactobacillus which are acid-tolerant anaerobes. Uses the same mechanism as in muscle tissue. This yields 2ATP/Glucose.

Question 30

Tell me about heterolactic fermentation

Answer 30

Occurs in heterofermentative lactic acid bacteria which catabolise sugars via the pentose phosphoketolase pathway (produces acetylCoA unlike the PPP).
A 5C sugar is split into G-3-P and Acetyl-P.
G-3-P is converted to lactate as in homolactic fermentation.
Acetyl-P is converted to ethanol.
Overall yield is 1ATP/Glucose.
See notes for actual pathway.

Question 31

Tell me about the Alcohol (ethanol) fermentation pathway

Answer 31

Glucose is converted to ethanol and CO2: Glucose to 2Pyruvate to 2Acetaldehyde to 2Ethanol.
Generates 2ATP/Glucose

Yeast and some bacteria including zymomonas mobilis generate pyruvate via the ED glycolytic pathway generating 1ATP/Glucose

Question 32

Tell me about mixed acid fermentation

Answer 32

Gram negative enterobacteriaceae such as E. coli convert pyruvate to a variable mix of end products including:
Ethanol, Formate, Acetate, H2, CO2, Lactate, Succinate
Yields 3ATP/Glucose
Check notes for pathway
Full pathway yields 2.3ATP/Glucose
Production of fumarate and succinate allows switching to anaerobic respiration.

Question 33

Tell me about ABE fermentation

Answer 33

Acetone, Butanol, Ethanol fermentation is carried out by gram positive clostridium species, produces solvents in the ratio of 3:6:1.
Used for the commercial production of acetone, there is renewed interest in butanol as a biofuel.
Generates AcetylCoA & ButyrylCoA which can be converted to ethanol and butanol to reform NAD+
Yields 3ATP/Glucose

Question 34

Tell me about malolactic fermentation

Answer 34

It is key in the secondary fermentation in wine, performed by lactic acid bacteria such as oenococcus oeni.
Malic acid is taken up and decarboxylated to produce lactic acid which is secreted. The antiport of Malic acid for Lactic acid by the protein MleP is key in PMF generation
ATP synthase is chemiosmotic, no need for redox balancing.