BIOC 2600 Workshop

Generation of ATP

Prof. DKY Shum

1. The figure to the right shows oxygen

consumption following addition of a
preparation of mitochondria to
-hydroxybutyrate dissolved in medium
containing 70 mM sucrose, 220 mM
mannitol, 2 mM HEPES buffer, 5 mM
magnesium chloride, 5 mM potassium
phosphate, 1 mM EDTA, and 0.1% bovine

(a)
(b)
(c)

(d)

serum album, pH 7.4 and at 25C. Dissolved

oxygen in the system is measured with an
oxygen electrode.

a. What sequence of reactions is initiated upon the addition of mitochondria to the system
and results in the rapid consumption of dissolved oxygen as shown in (a) of the figure?
b. What accounts for the slow rate of oxygen consumption observed in (b) of the figure?
c. Suggest events that lead to the rapid oxygen consumption upon addition of 500 nmol of
ADP to the system (part (c) of the figure).
d. What accounts for the slow rate of oxygen consumption observed in (d) of the figure?
e. In what form is oxygen consumed in parts (a) to (d) of the figure?
f. How many moles of ATP are synthesized per oxygen atom consumed?

2. The following table shows experimental findings of oxygen consumption during the
oxidation of endogenous carbohydrates in minced pigeon breast muscle preparations as
by Hans Krebs (1937).
Oxygen consumption (mmol)
Time (minutes)
30
60
90
150

No citrate added

3 mmol citrate added 3 Difference

29
47
51
53

31
68
87
93

2
21
36
40

a. If citrate (C6H8O7) were completely oxidized to CO2 and H2O as in a balanced

chemical reaction, how many molecules of O2 would be consumed per molecule of

citrate? How does this value differ from the experimental observation?
b. Why is the consumption of O2 low (5 mmol) in the presence of arsenite and malonate?
If citrate were oxidized to -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?

3. Peter Mitchell hypothesized that oxidative phosphorylation in mitochondria involves

formation of a proton gradient which drives the synthesis of ATP. To test the hypothesis,
inside-out submitochondrial vesicles have been prepared. This involves the isolation of
mitochondria by differential/gradient centrifugation and then stripping off the outer
membrane. Then, ultrasound is used to produce inside-out mitochondrial vesicles.
a. Draw a sketch of the inside-out mitochondrial vesicles.
b. Mark in the sketch the direction in which are protons are pumped.
c. The addition of dinitrophenol, a proton ionophore, does not compromise the activity
of the electron transport chain but does decrease oxidative phosphorylation. Why?
d. The addition of sodium azide, an electron transport inhibitor, decreases oxidative
phosphorylation in the inside-out vesicles. Why?
e. Oligomycin causes an increase in proton-motive force in the inside-out vesicles, yet
oxidative phosphorylation is inhibited. Why?
f. When the inside-out vesicle preparation is incubated with chloramphenicol, oxidative
phosphorylation does not appear to be affected. Why?
g. Treatment of the inside-out vesicle preparation with sodium dodecyl sulfate causes
decrease in oxidative phosphorylation. Why?
h. Mechanical disruption of inside-out vesicles can lead to dissociated F1 particles that
are no longer associated with the F0. In this case, electron transport continues but no
ATP is synthesized. Explain.
i. When the F1 particle is removed from the F0 but the F0 remains in the membrane of
the inside-out particles, the proton-motive force dissipates but the electron tranposrt
system continues. The outside medium becomes acidic. What can you infer about
the function of the F0?