Week 10 Hitchcock Lecture 2

Question 1

What are the metabolic modes of E. coli?

Answer 1

3 modes:

• Aerobic respiration
• Anaerobic respiration
• Fermentation

Question 2

What is anaerobic respiration?

-What replaces the oxygen?

Answer 2

the use of an electron transport chain to generate a PMF but with a terminal acceptor that is not oxygen
-The membrane bound respiratory electron transport chain uses an inorganic or organic terminal acceptor other than oxygen. Because oxygen is such a good electron acceptor (midpoint potential of O2/H20 couple = +820 mV), alternative acceptors release less energy per oxidised molecule

Question 3

Does glycolysis still occur at anaerobic conditions?
What happens to the pyruvate after?
What happens to the products produced; formate and acetyl-CoA?

Answer 3

The formation of 2 pyruvates from glucose still occurs as it does not require oxygen.

• E. coli uses a different enzyme called pyruvate-formate lyase, or PFL, which cleaves pyruvate into Acetyl-CoA and formate
• Formate, which is an excellent electron donor, is oxidised by the anaerobic respiratory chain. acetyl-CoA is reduced, to acetate, which is subsequently excreted from the cell as an overflow metabolite in order to balance carbon flux. This process involves the enzymes phosphotransacetylase (Pta) and acetate kinase (Ack) and allows generation of some extra ATP by additional substrate level phosphorylation

Question 4

What happens to the krebs cycle under anaerobic conditions?

Answer 4

acetyl-CoA being combined with oxaloacetate to make citrate, but this oxidative branch is repressed and is only functional as far as alpha-ketoglutarate. In the absence of oxygen the alpha-ketoglutarate DH enzyme is inhibited and so the cycle can’t continue in the clockwise forward direction. And metabolic flow in the C4 section of the pathway, so between succinyl-CoA and oxaloacetate, is reversed. This reductive branch offers the possibility of energy conservation by reduction of fumarate to succinate by this enzyme fumarate reductase (FRD). Here flux into the cycle is less, with much of the acetyl-CoA converted to acetate to generate extra ATP by substrate level phosphorylation.

Question 5

What is the general rule in electron transport chains?

Answer 5

Electron donors are the lowest values and the electron acceptor has the highest mV value. The quinone sits in between them

Question 6

What is the enzyme responsible for converting formate to CO2?
-What is formate dehydrogenase?

Answer 6

is the formate dehydrogenase or Fdn, and contains G, H and I subunits
-Fdn is membrane anchored and periplasmic facing and the electrons released from the oxidation of formate pass through a wire of redox cofactors in the protein, and reduce menaquinone to menaquinol. The protons are released from formate into the periplasm, and those taken up upon MQ reduction are from the cytoplasm, so this generates a redox loop that contributes to the PMF with a proton per electron ratio of 1

Question 7

What is Nar?

Answer 7

Nar is also a multi-subunit enzyme anchored in the cytoplasmic membrane but faces the cytoplasmic side of the membrane. Electrons from menaquinol pass through another wire of redox cofactors and are used to reduce nitrate, NO3-, to nitrite, NO2-. Here we have another redox loop – so protons are released in the periplasm when menaquinol is oxidised and used up in the cytoplasm upon nitrate reduction

Question 8

What is Nap?

-Since Nap generates less energy than Nar, why is it used?

Answer 8

Nitrate reductase (NapABC) is a multi-subunit enzyme containing two Mo[MGD] cofactors, an [Fe-S] cluster and 6 c-type cytochromes. a second type of nitrate reductase in E. coli called Nap, which faces the periplasm, remember Nar was facing the cytoplasm, but in Nap the catalytic domains are in the periplasm. This enzyme is not energy conserving as the protons released when menaquinol is oxidised to menaquinol and those taken up when nitrate is reduced to nitrite are from the same side of the membrane, the periplasm, thus a PMF is not generated
-Nap functions at lower nitrate concentrations than Nar, and does not require nitrate uptake into the cytoplasm, and may be important to dispose of excess electrons during growth on very reduced carbon sources.

Question 9

What determines the proton translocating capacity of an electron transport chain?

Answer 9

donor/acceptor couples redox potentials and the organisation of the enzymes in the membrane, which alters how electron transfer is coupled to proton translocation

Question 10

What is Frd?

Answer 10

Fumarate reductase; is a multi-subunit enzyme containing a flavin cofactor and three FeS centres