Question

In 1937 Hans Krebs deduced the operation of the citric acid cycle from careful observations on the oxidation of carbon compounds in minced preparations of pigeon flight muscle. (Pigeon breast is a rich source of mitochondria, but the function of mitochondria was unknown at the time.) The consumption of O2 and the production of CO2 were monitored with a manometer, which measure changes in volume of a closed system at a constant pressure and temperature. Standard chemical methods were used to determine the concentrations of key metabolites.

(Remember, radioactive isotopes were not available then.)
In one set of experiments Krebs measured the rate of consumption of O2 during the oxidation of endogenous carbohydrates in the presence or absence of citrate. As shown in Table 1, addition of a small amount of citrate resulted in a large increase in the consumption of oxygen. Szent-Gyorgyi (1925) and Stare and Baumann (1936) had previously shown that fumarate, oxaloacetate, and succinate also stimulated respiration in extracts of pigeon breast muscle.

Table 1: Respiration in minced pigeon breast in the presence and absence of citrate Oxygen Consumption (mmol) Time (minutes) No citrate 3 mmol citrate Difference 30 29 31 2 60 47 68 21 90 51 87 36 150 53 93 40

When metabolic poisons, such as arsenite or malonate (whose modes of action were undefined), were added to the minced muscles, the results were much different. In the presence of arsenite, 5.5 mmol of citrate were converted into about 5mmol of α- ketoglutarate. In the presence of malonate an equivalent conversion of citrate into succinate occurred. Furthermore, in the presence of malonate roughly 5mmol of oxygen were consumed (above background levels in the absence of citrate), which was twice as much as in the presence of arsenite.
Finally, Krebs showed that the minced muscles were actually capable of synthesizing citrate if oxaloacetate was added and all traces of oxygen were excluded.
None of the other intermediates in the cycle led to a net synthesis of citrate in the absence of oxygen.
1). If citrate (C6H5O7) were completely oxidized to CO2 and H2O, how many molecules of O2would be consumed per molecule of citrate? What is it about the results in Table 1 that caught Krebs's attention? (5 pts)

2) Why is the consumption of oxygen so low in the presence of arsenite and malonate? If citrate were oxidized to α-ketoglutarate (C5H6O5), how much oxygen would be consumed per molecule of citrate? If citrate were oxidized to succinate (C4H6O4), how much oxygen would be consumed per molecule of citrate? Does the observed stoichiometry agree with the expectations based on these calculations? (5 pts)

3) Why in the absence of oxygen does oxaloacetate alone cause an accumulation of citrate? Would any of the other intermediates in the cycle cause an accumulation of citrate in the presence of oxygen? (5 pts)

4) Toward the end of the paper Krebs states, "While the citric acid cycle thus seems to occur generally in animal tissues, it does not exist in yeast or in E. coli, for yeast and E. coli do not oxidize citric acid at an appreciable rate." Why do you suppose Krebs got this point wrong? (5 pts)

Answer 1

1. Citrate require 4.5 moles of oxygen per citrate molecule

C6H8O3 + 4,5 0, → 6 CO2 + 4H2O

Kerb noticed that in presence of citrate more oxygne is consumed. Citrate leads to higher consumption of O₂than what is required for its complete oxidation. Hence, it can be said that citrate facilitated O₂consumption.

2. Arsenate and malonate production of reactive oxygen species (ROS) which in turn leads to cellular and metabolic impairment through oxidative damage and hence reduce the consumption of oxygen

One oxygen molecule is required in conversion of citrate (C6) to alpha ketoglutarate (C5), one CO2 molecule is released.

If citrate (C6) is converted to succinate (C4), 2 oxygen (O2) molecule are required.

Yes, the observed stoichiometry agrees with the expectations based on these calculations

3. Oxaloacetate leads to citrate formation even when it is not in enough amount. The only impact of low Oxaloacetate would be lower rate of acetyl CoA metabolism and ATP formation.

It is also accumulated when kinase phosphorylates

4. Kerb cycle can take place in absence of oxygen and this might be a worng validation for saying that kerb cycel does not occur in yeast and E coli.