BIOCHEMISTRY II

Electron transport and oxidative phosphorylation

1. Dinitrophenol , an uncoupler of oxidative phosphorylation (now considered to be

extremely to be toxic for clinical use), was formely used as a weight reducer.
Explain how it was effective.

• Dinitrophenol

o It is uncoupler of oxidative phosphorylation

o Used as weight reducer

o Properties :

 A lipophilic proton carrier that readily diffuses through the

mitochondrial membrane

o Causes :

 Collapse the proton gradient without generating ATP

 Proceed at a rapid rate without establishing a proton gradient

o Consequence :

 Release as heat rather than being used to synthase ATP

 Becomes toxic as it take in high doses and person wil be

skinner

2. Each of threee Warburg vessels (A, B and C) contained :

• Intact mitochondria

• α-ketoglutarate

• ADP

• Hexokinase

• Glucose

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 • Buffer

To vessel B, 10-3 M amytal was added. To vessel C, 10-5 Dinitrophenol (an

uncoupler of oxidative phosphorylation) was added. After 30 min vessel A
showed an uptake of 224 mm3 O2 and analysis of the contents showed
accumulation of 50 µmoles of glucose-6-phosphate.

Explain why similar or different values would be expected for vessel B and C.

Vessel A

• Normal

• Formation G6P

• Consumation O2 (uptake)

Vessel B

• Addding of Amytal

o Properties

 Site-specific inhibitors of electron transport

o Blocks complex I

o Consequence

 Level O2 drops (Took measure O2)

 Prevent the passage of electrons by binding to a component

of the chain

 No respiration

 No oxidative phosphorylation

 No Glucose-6-phosphate

Vessel C
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 • Adding dinitrophenol

o Consequence

 No ATP

 No formation of Glucose-6-phosphate

 O2 uptake

Properties (function)

• Intact mitochondria

o Begun process in electron transport system

• α- ketoglutarate

o Oxidation to form NADH

• ADP / ATP

o Contribute free energy

• Hexokinase

o An enzyme involve in glycolysis process

• Inorganic phosphate

o Bind to ADP to formed ATP

• Glucose

o Precursor in glycolysis process

• Buffer

o To make condition in physiological environment

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3. Oxygen uptake was measured in each of three Warburg flasks that contained the
following :

• Rat liver mitochondria

• 21 µmoles of α-ketoglutarate

• 30 µmoles of phosphate

• Catalytic amounts of ATP

• Cytochrome C

• MgSO4

Flasks B and C also contained yeast hexokinase and glucose (35 µmoles).
Dinitrophenol (3X10-5) was added to flask C only. The following values

Were obtained for the various flasks.

• Plot a graph (oxygen uptake versus time)

• Explain the differences in rates of respiration

o Flask A

 Less O2 uptake

 Basal level of respiration

 Resting phase

 No demand of ATP

 Oxidative phosphorylation at normal rate

 No additional energy

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  Prolong time

o Flask B
Hexokinase

 Glucose Glucose-6-phosphate

ATP ADP

 O2 uptake increase because of added of susbstrate

 Respiration higher than normal

 O2 consumption rate higher

 More ATP produce by glycolysis process

o Flask C

 Contain Dinitrophenol

 Types of inhibitor to ATP formation

 Uncoupler (ET – Phos)

 Anti-coupling agent

 Increase O2 uptake (Rapid O2 consumption)

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  Increase permeability of inner mitochondrial (Difuses
through)

 Consequences :

 Liver is demand for ATP for respiration

o Properties (function)

 Glucose

 Create demand for ATP

 Hexokinase

 Convert glucose to Glucose-6-phosphate

 MgSO4

 Activated ATP (metalic ion to activate hexokinase)

 Dinitrophenol

 Uncoupler (uncoupler electron transport chain from

oxidative phosphorylation)

 ATP

 Contribute to free energy

• What can you do to test your explanation ?

o Add glucose and hexokinase into flask C

o Add DNP into flask A

• How efficient is the mitochondrial preparation with respect to ATP

formation ? (1 µmole of O2 occupies 22.4 mm3). Explain your answer.

Efficiency

o Glucose + O2 ---- ATP + CO2 + H2O

o 1 µmol O2 = 22.4 mm3

o 2 µmol O = 22.4 mm3

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  X = 120 mm3 (From the graph)

 X = 120 / 22.4 X 2

 10.71 µmol of O

Phosphate 30µmole

o P = 30 µmol ; O= 10.71 µmol

o P/O = 30/10.71

o P/O = 2.8

o P.O = 2.8/3 X 100

o 93.3%

4. Fresh prepared rat liver mitochondria were incubated in air in a buffered isotonic
medium containing the following components :

• Succinate

• Inorganic phosphate (Pi)

• ADP

• Glucose

• Hexokinase + Mg2+

The following results on oxygen consumption and Pi uptake were obtained :

O2 Consumed (µmoles) : 1.3

Pi disappeared (µmoles) : 5.1

When KCN and Potassium ferricyanide were added to similar suspensions, with
and without antimycin A, the following data were obtained :

Antimycin (-) Antimycin (+)

O2 consumed (µmoles) 0 0

K3Fe(CN)6 reduced (µmoles) 5.1 0.1

Pi disappeared (µmoles) 2.5 0

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• Explain the role of each added component (those that are underlined) in
the various mixtures.

o Succinate

• To activate succinate dehydrogenase (Succinate to fumarate)

• Electron received

o Inorganic phosphate /ADP

• Produce ATP

o Glucose

• Energy yield

o Hexokinase + Mg2+

• Hexokinase (An enzyme involve in glycolysis process)

• Mg2+ (Required for the activity of enzymes especially in

oxidative phosphorylation)

o KCN

• A poisonous compound

• Blocked complex IV in electron transport system

o K3Fe(CN)6

• Called the Drabkin’s solution

• As electron acceptor

• Artificial of complex IV in electron transport system

o Antimycin
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 • A crystalline antibiotic active against various fungi , insects and
mites

• Antimycin blocks the flow of electrons from semiquinone to

ubiquinone in the Q-cycle of complex III in oxidative
phosphorylation.

• It inhibits the electron transport pathway thus preventing the

consumption of oxygen (which occurs at Complex IV) and
disrupting the proton gradient across the inner membrane.

• The disruption of the proton gradient that prevents the

production of ATP as protons are unable to flow through the ATP
synthase complex

• Discuss the nnumerical values that were obtained. Include calculation

on efficiency of phosphorylation and discussion of energy conservation
sites.

o 1 µmole of O2 = 22.4 mm3

125 mm3

o 1.3 O2 = 1.3 X 2

2.6 µmol O

o P/O ratio = 5.1/2.6

1.96 near to 2 ATP

Efficiency

o 1.96/2 X 100

98.1%

Tubes with no Antimycin

o P/O ratio 1 mol O2 = 2 atom (ATP)

o K3Fe(CN)6 = 5.1/2

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 2.55 oxygen

o P/O ratio = 2.5/2.55

0.980 near to 1 ATP

Tubes with Antimycin

o K3Fe(CN)6 = 0.1/2

0.05 Oxygen

o P/O ratio = 0/0.05

O ATP

5. A type of adipose tissue, found prominently in infants but little in adults, has a
high content of mitochondria, giving it a brown appearance. These mitochondria
have a P:O ration of less than 1.

• What physiological function might these mitochondria perform?

To produce more energy require for infant growth

• What differences might be found between the mitochondria of brown

fat and those of liver to account for their altered function ?

Adipose tissue brown (http://www.ncbi.nlm.nih.gov/pubmed/14715917)

o Thermogenic type of adipose tissue containing a dark pigment

o Purpose is to generate body heat

o Arise during embryonic life

o Contains more capillaries since it has a greater need for oxygen

than most tissues

Adipose tissue white

(http://journals.cambridge.org/action/displayAbstract?fromPage=online&ai
d=804764)
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o Adipose tissue comprisingly the bulk of the body fat

o Contain a single large fat droplet, which has forced the nucleus to
be squeezed into a thin rim at the periphery

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