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OUTLINE
I. Mitochondria
II. Respiratory Chain and Oxidative Phosphorylation
A. Respiratory Chain
B. Oxidative Phosphorylation at the Respiratory Chain
III. Oxidative Phosphorylation
IV. ATP Synthesis
A. Proton Translocation and ATP Synthesis
B. ATP Synthase and Couplers
V. Respiratory Control
VI. Substrate Shuttles
VII. Clinical Correlations
VIII. Summary
IX. Competencies
X. Sample problems
OBJECTIVES
At the end of the lecture, the student should be able to:
1. To develop understanding of the structural organization of the
mitochondria;
2. To develop an understanding of the coupling mechanism
between oxidation of reducing equivalents and ATP synthesis;
and
3. To identify poisons/toxins that block the Electron Transport
Chain (ETC) and ATP synthesis
References:
Harpers Illustrated Biochemistry
Transes from previous batches
Legend: Italicized quoted from the lecturer; bold emphasis, or
from references
I. MITOCHONDRIA
- Mitochondria have an outer membrane which is permeable
to most molecules and inner membrane which is selectively
permeable, enclosing a matrix within. Inner membrane is not
permeable to ionized molecules.
Review: Enzymes in the Citric Acid Cycle that will form NADH
include
Pyruvate
dehydrogenase,
Isocitrate
dehydrogenase, -ketoglutarate dehydrogenase, and
malate dehydrogenase. While for FADH, the enzyme is
Succinate dehydrogenase. NADH and FADH store the
energy that is released so that the energy is not wasted; rather
they are transported to electron chain and converted to ATP.
NADH and FADH represent the reducing equivalents, brought
to ETC to oxidize bringing in the oxygen, which will be reduced
to water.
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Figure 5. Coenzyme Q.
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Figure 8. Complex IV
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3. Hydroxyperoxidases
Use oxygen as hydrogen peroxide or organic peroxide
as substrate
Protects the body against harmful peroxides
4. Oxygenases
Catalyze direct transfer
Incorporation of oxygen into a substrate
Figure 12. Complexes I, II, III, and IV acts as proton pumps. (Harpers, p.
126)
Figure 11. Overview of the electron flow through the respiratory chain.
(Harpers p. 123)
F0 subcomplex
- Spans the membrane
- Consists of several C protein subunits which form a disk
- Forms a proton channel
- Protons re-enter the membrane via F0, causing it (and the
attached subunit) to rotate to the right, driving the
production of ATP in the F1 complex
Binding Change Mechanism
- The conformation of the -subunits in F1 is changed as the
axis rotates from one that binds ATP tightly to one that
releases ATP and binds ADP and Pi so that the next ATP
can be formed
- F1 is squeezed causing the release of ATP; portion of the
membrane contains ADP and Pi, with the action of ATP
synthase ATP is produced
Uncouplers
- Increases membrane permeability to ions
- Collapses proton gradient
- Allows H+ to pass without going through the ATP synthase
- Results in the flow of uncouple electron through respiratory
complexes from ATP synthesis
- [4] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano
POISON
SITE OF ACTION
Inhibitors of the Respiratory Chain
Barbiturates
Complex I
Antimycin A
Complex III
Dimercaprol
H2S
Complex IV
Carbon Monoxide
Cyanide
Malonate
Complex 2
Inhibitors of Oxidative Phosphorylation
Atractyloside
Oxidative Phosphorylation
Oligomycin
Halts
both
Oxidation
and
Phosphorylation
Uncouplers:
Halts coupling of oxidation and
2, 4-dinitrophenol
phosphorylation
Thermogensin (protein
in
brown
adipose
tissue)
V. RESPIRATORY CONTROL
- Because oxidation and phosphorylation are tightly coupled,
the rate of respiration of mitochondria can be controlled by
the availability of ADP.
- Most cells in the resting state are in State 4.
STATE
State 1
State 2
State 3
State 4
State 5
Inhibitors of Oxidation-Phosphorylation
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How It Works:
NADH reduces oxaloacetate to form malate in the
intermembrane space by malate dehydrogenase
ii. Malate enters the matrix via the Malate- -ketoglutarate
transporter
iii. Malate reduces NAD+ to form NAD + H+ and oxaloacetate
in the matrix by malate dehydrogenase
iv. Oxaloacetate is transaminated by glutamate to form
Aspartate
and
-ketoglutarate
by
aspartate
aminotransferase
v. Aspartate leaves the matrix via Glutamate-aspartate
transporter
vi. Aspartate is deaminated by -ketoglutarate forming
oxaloacetate and glutamine
i.
B. Glycerol-3-Phosphate Shuttle
- NADH binds with Complex II
- ATP produced: 2 (but in actuality, 1.5)
- Occurs in brain and white muscle
- Deficient in heart
How it works:
Dihydroxyacetone phosphate is reduced by NADH + H+ in
the cytosol to form glycerol 3-phosphate. The reaction is
catalyzed by cytosolic glycerol 3-phosphate dehydrogenase
ii. Mitochondrial
glycerol
3-phosphate
dehydrogenase
(reduction) transfers the electrons from glycerol 3-phosphate
to FAD to form FADH2 and dihydroxyacetone phosphate
iii. The electrons are then transferred to ubiquinone
i.
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11.
12.
13.
14.
15.
16.
X. SAMPLE QUESTIONS
Identify the different layers of the mitochondrial membrane and the enzymes that they contain.
In applying your knowledge of the process of Respiratory Chain, arrange its components with regards to the electron flow. Complex
I, Complex II, Complex III, Complex IV, Q, cyt c: ___/___>___>___>___>___
Match Flavoprotein and Fe-S to their respective complexes.
It states that the electrochemical potential difference from the asymmetric distribution of the H+ drives the mechanism for ATP
formation.
Where is respiratory chain embedded?
In what complex in ETC catalyzes electron transfer from FADH2 to Q.
What mechanism does the ATP synthase utilize to produce ATP?
How does barbituates affect inhibit NAD-linked dehydrogenases?
How does malonate affect the function of succinate dehydrogenase?
Arrange the following to the right sequence of ATP production by ATP synthase.
a. F0 and the bent axle rotate counter-clockwise
b. H+ crosses the membrane into the matrix via F0
c. B subunits bind ADP+Pi forming the next ATP
d. F1 squeezed, causing change in conformation of B subunits
e. ATP released
What are the end products of respiratory chain?
T/F. Aerobic organisms are able to capture far greater proportion of available free energy of respiratory substrates than anaerobic
organisms.
How many ATPs are produced per revolution of ATP synthase?
T/F. Ubiquinone and cytochrome are embedded in the inner membrane of the Mitochondria
Sites electrons pass before going to Q from complex 1.
Which Fe-S is involved in the transport of electrons from Qh2 to cytochrome c?
Answers:
1. See Page 1 of trans
2. I/II>Q>III>cyt c>IV
3. I,II/I,II,III
4. Chemiosmotic Theory
5. Inner Mitochondrial Membrane
6. Complex II
7. Binding Change mechanism
8. Blocking transfer from FEs to Q in complex I & preventing transfer of electrons
9. Through competetive inhibition
10. b>a>d>e>c
11. Water and ATP
12. T
13. 3
14. F, they are free flowing
15. FMN, Fe-S
16. Rieske FE-s
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