[BIOCHEMISTRY] 2.

5 Electron Transport Chain and Oxidative Phosphorylation

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD
Dr. Balcueva
7 August 2013
14 Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

OUTLINE

Enzymes in the Outer Membrane

1. Acyl Coenzyme A synthetase
2. Glycerolphosphate acyl transferase

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

*Same enzyme located in the cytosol

Enzymes in the Inner Membrane

1. Electron carriers 4 complexes responsible for oxidizing
the different reducing coenzymes
2. ATP synthase
3. Membrane transporters
4. Cardiolipin a phospholipid that is concentrated in the
inner membrane together with the enzymes of the
respiratory chain, ATP synthase, and various membrane
transporters (Harpers Chapter 13, p. 122).
Enzymes in the Mitochondrial Matrix
1. Citric Acid Cycle
2. Pyruvate dehydrogenase produces NADH
3. -oxidation enzymes

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.

Figure 2. Structure of the mitochondrial membranes. (Harpers p. 122)

II. RESPIRATORY CHAIN AND OXIDATIVE

PHOSPHORYLATION
- Mitochondria contains the respiratory chain that collects and
transports reducing equivalents such as NADH and FADH (H or electrons) to oxygen
- Oxygen oxidizes reducing equivalents to form water at the
end of the chain
- Oxidation is coupled to ATP formation from ADP and
inorganic phosphate (Pi)
A. Respiratory Chain
- H+ and electrons flow through the respiratory chain in order
of decreasing redox potential (NADH = -0.32 volts (highest
redox potential); fumarate/succinate = +0.8 volts)
- In reactions involving oxidation-reduction, the ability of the
reactants to donate or accept electrons is proportionate to
free energy (redox potential)
- Redox potential of a system is compared to redox potential
of a hydrogen electrode at -0.42 volts

Figure 1. Role of the respiratory chain of mitochondria in the conversion of

food energy to ATP. (Harpers p. 123)

- [1] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

- Consists of redox carriers that start with NAD/NADH to
O2/2H2O
- Fumarate/succinate bypass NAD/NADH and are linked to
flavoprotein dehydrogenases
- Ubiquinone (Q):
Structure similar to vitamin K and E;
Mobile component of the respiratory chain
Collects reducing equivalents from flavoprotein

Complex I (NADH-Q oxidoreductase)

Table 1. Standard Reduction Potentials of Respiratory Chain and Related

Electron Carriers.

Figure 4. Complex I of Respiratory Chain.

NADH + Q + 5H+ NAD + QH2 + 4H+

- Catalyzes electron transfer from NADH to Q, coupled with

+
the transfer of 4 H across the membrane
- Electrons are transferred from NADH to FMN initially, then to
a series of Fe-S centers and finally to Q. (Harper)
- 4 protons produced
- Flow of e : NADH FMN Fe-S centers Q

Overview of the Process of electron flow via Respiratory

Chain
- Electrons flow through the Respiratory Chain via redox
carriers that start from NAD/NADH to O2/2H2O through a
redox span of 1.1 V (Harpers)
- Electrons Passes through three Large Protein Complexes:
1. Complex I (NADH-Q oxidoreductase)
Electrons transferred from NADH Q
2. Complex III (Q-cytochrome c oxidoreductase)
passes the electrons from Q cyt c
3. Complex IV (cytochrome c oxidase)
Completes the chain
Passing the electrons to O2, reducing it to water
- Fumarate/succinate has a more positive redox potential than
NAD/NADH.
Gives electrons ability to bypass the utilization of
NAD/NADH and Complex I
Thus, Electrons pass directly to Q via Complex II
(Succinate-Q reductase )

Figure 5. Coenzyme Q.

Complex II (Succinate-Q reductase)

- FADH2 is formed during the conversion of succinate to
fumarate in the citric acid cycle and electrons are passed via
several Fe-S centers to Q.
- Flow of e : FAD (from succinate) Fe-S centers Q

B. Oxidative Phosphorylation at the Respiratory Chain

- NADH 3 moles of Pi are incorporated to 3 ADPs
- FADH 2 moles of Pi are incorporated to 2 ADPs
- P:O = 3
- Free energy from glucose through substrate phosphorylation
(103kJ/mol glucose)
- Free energy through respiratory chain (68%)

Figure 6. Coenzyme Q and Complex III.

Figure 7. Succinate/Fumarate, CoQ, and Complexes II and III.

Figure 3. Oxidative Phosphorylation at the Respiratory Chain. (Harpers p.

124)

- [2] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

May contain one, two, or four Fe atoms via cysteinethiol (SH) groups except in complex III, where Fe is
linked to a histidine-SH group (Rieske Fe-S)

Complex III (Q-cytochrome c oxidoreductase)

QH2 + 2 Cytc(o) + 2H (matrix) Q + 2 Cytc(r) + 4H

- This process is believed to involve cytochromes c1, bL and bh,

and a Rieske Fe-S (an unusual Fe-S in which one of the Fe
atoms is linked to two histidine residues rather than two
cysteine residues) (Harper).
- aka the Q cycle
Q exists in 3 forms: Oxidized quinone (Q), Reduced
quinol (QH2), and Semiquinone (Harper)
- Oxidation of QH2 to Q
1st e is donated to cyt c via a Rieske Fe-S and cyt c1
2nd e is donated to a Q to form the semiquinone via cyt
bL and cyt bH
+
2 H are released into the intermembrane space
- Oxidation of a second QH2 to Q
1st e is donated to cyt c via a Rieske Fe-S and cyt c1
2nd e is donated to the semiquinone, reducing it to QH2
+
Another 2 H are released into the intermembrane
space
+
2 H are taken up into the inner mitochondrial
membrane from the matrix
- 4 protons produced
- Flow of e : QH2 Rieske Fe-S cytochrome c

Figure 9. Fe-S proteins. (Harpers p. 124)

III. OXIDATIVE PHOSPHORYLATION

Chemiosmotic Theory
- Peter Mitchell (1961) proposed mechanism for oxidative
phosphorylation
- The H+ ion transfer results in an increase in the H+ ion
concentration in the space between the inner and outer
mitochondrial membranes. It is necessary to move H+ ions
back across the membrane. This transfer of H+ ions is
necessary in the synthesis of ATP.
- It assumes that the H+ ion gradient is a significant factor
promoting the conversion of ADP to ATP, occurring in the
four complexes present in the inter mitochondrial membrane
result in a net transfer of H+ ions across the membrane.
- Energy from oxidation of components in the respiratory
chain is coupled to the translocation of hydrogen ions from
the inside to the outside of the inter mitochondrial membrane
space.

Figure 8. Complex IV

Complex IV (Cytochrome c oxidase)

4 Cytc(r) + O2 + 8H 4 Cytc(o) + 2 H2O + 4H
-

- Transfer of 4 e from Cyt c to O2 involves two heme groups,

a and a3, and Cu
- Electrons are passed initially to a Cu center (CuA).
- CuA contains 2 Cu atoms linked to 2 protein Cysteine-SH
groups
- A second Cu center, CuB, is linked to heme a3
+
- Of the 8 H removed from the matrix,
o 4 are used to form 2 H2O
o 4 are pumped into the intermembrane space
- Thus, for every pair of e- passing down the chain from
+
NADH or FADH2, 2 H are pumped across the membrane by
Complex IV
- O2 remains tightly bound to Complex IV until it is fully
reduced
- This minimizes the release of potentially damaging
intermediates, such as superoxide anions or peroxide, which
are formed when O2 accepts 1 or 2 e , respectively
+
- 2 protons produced. Based on the reaction given above, 4H
were released in the intermembrane space. This is because
+
the 4H released reacted with O2 forming water.
Flow of e : Cyt c CuA heme a heme a3 and CuB O2

Figure 10. Electrochemical gradient.

Enzymes Involved in Oxidation-Reduction Reaction

(Oxidoreductases)
1. Oxidases
Remove hydrogen from a substrate using oxygen as an
acceptor
Forms water or hydrogen peroxide
a. Cytochrome oxidase
o Located in Complex IV
o Has a heme prosthetic group
o Terminal component of the chain
o Carries electron to oxygen (tightly bound to
Complex IV)
a. Flavoproteins contain flavin adenine dinucleotide
(FAD)

Components of the Respiratory Chain Complexes

1. Flavoproteins: Complexes I and II
a. FMN FMNH2
b. FAD FADH2
2. Fe-S: Complexes I, II, and III

- [3] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

2. Dehydrogenases
Transfer hydrogen from one substrate to another
Cannot use O2 as a hydrogen acceptor specific for the
substrate; uses coenzymes or hydrogen acceptors
Example: NAD and FAD dehydrogenase

B. ATP Synthase and Couplers

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)

ATP Synthase Complex

- also called Complex V
- Driven by the proton motive force
- Functions as a rotary motor to form ATP in the presence of
Pi + ADP
- It is composed of two parts: F0 and F1
- F1 for binding of ATP & ADP
- F0 for translocation of protons
F1 subcomplex
- Projects into the matrix
- Contains the phosphorylation mechanism
- Attached to F0 through the subunit, which is in the form of
a bent axle
- Consists of 3 and 3 subunits attached around the
subunit. The and subunits are fixed into the membrane
and do not rotate with the subunit

Figure 11. Overview of the electron flow through the respiratory chain.
(Harpers p. 123)

IV. ATP SYNTHESIS

A. Proton Translocation and ATP Synthesis
- Complex I, III and IV acts as proton pumps, creating a
proton gradient across the membrane negative on the matrix
side
- Complex I and III translocate 4 protons each
- Complex IV translocate 2 protons
- H ions enter or pass across the inner mitochondrial
membrane thru the ATP synthase w/ ATP synthesis
- ATP cannot be generated without the protons which are
transferred into the inter membrane space
- ATP goes out in exchange of ADP: anti-port
- Electron chemical gradient gives rise to Proton Motive Force

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

Proton Motive Force

- Drives ATP synthesis as proton flow back into the matrix
with the help of ATP synthase enzyme
- Caused by the electrochemical potential difference
- Protons accumulate in the intermembrane space because
the inner mitochondrial membrane is impermeable to ions
- Thus, negative () on the matrix side
- Recall: Complexes I, III, and IV act as proton pumps
- Protons from the matrix across inner mitochondrial
membrane into the intermembrane space

Mechanism of ATP Production by ATP Synthase

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

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

- Malonate is a competitive inhibitor of succinate
dehydrogenase in Complex II. (Also carboxin and
thenoyltrifluoroacetone. All with succinate as substrate.
- Antimycin A, BAL and dimercaprol inhibits between
Cytochrome b and c in Complex III by blocking electron
transfer from cytochrome heme bH to ubiquinone.
- Others such as myxothiazol and stigmatellin inhibit
transfer through complex III by binding to the Q0 site and
blocking transfer of electrons from ubiquinol.
- H2S, carbon monoxide and cyanide inhibits cytochrome
oxidase in Complex IV and can therefore totally arrest
respiratory chain.
- The antibiotic oligomycin completely blocks oxidation and
phosphorylation.
- Uncouplers (e.g. 2,4- dinitrophenol) dissociate oxidation in
the respiratory chain from phosphorylation. It causes
respiration to be uncontrolled, in vivo, since the rate is no
longer limited by the concentration of ADP and Pi.
Thermogenin (the uncoupling protein) is a physiologic
uncoupler found in brown tissue that functions to generate
body heat, particularly for the newborn and during
hibernation.
- Atractyloside inhibits oxidative phosphorylation by inhibiting
the transporter of ADP into and ATP out of the
mitochondrion.

Alternating alpha and beta subunits form knob of F 1

Protons flow through membrane
First bind to amino acid in Fo
Protons then bind to amino acid residue on C subunit
Gamma and epsilon units rotate
Gamma subunit movement cause change in beta subunit
A and B units of Fo + delta subunit of F1 = stator to hold
alpha and beta subunits in place
- Gamma and c subunits form the rotor

Table 2. Summary of Inhibitors of the Respiratory Chain and Oxidative

Phosphorylation

Figure 14. Exchange diffusion systems. (Harpers, p. 129)

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

CONDITIONS LIMITING RATE

OF RESPIRATION
Availability of ADP and substrate
Availability of substrate only
Availability of ADP only
Capacity of the respiratory chain
Availability of oxygen only

Inhibitors of Oxidation-Phosphorylation

VI. SUBSTRATE SHUTTLES

- NADH cannot penetrate the mitochondrial membrane, but it
is produced continuously in the cytosol by 3phosphoglyceraldehyde dehydrogensase (enzyme in
glycolysis).
- However, under aerobic conditions, extramitochondrial
NADH does not accumulate and is presumed to be oxidized
by the respiratory chain in mitochondria.
- The transfer of reducing equivalents through the
mitochondrial
membrane
requires
substrate
pairs
(SHUTTLES), linked by suitable dehydrogensases on each
side of the mitochondrial membrane.
A. Malate-Aspartate Shuttle
- NADH binds with Complex I
- Found in liver, kidney and heart mitochondria
- ATP produced: 3 (but in actuality 2.5)
- Occurs in HKL heart, kidney, liver
- Universally used shuttle

Figure 15. Sites of inhibition of the respiratory chain by specific drugs,

chemicals, and antibiotics. (Harpers, p. 128)

- Barbiturates like amobarbital ,piercidin & amytal inhibit

NAD-linked Dehydrogenase by blocking the transfer from
FeS to Q in Complex I by preventing transfer of electrons
from iron sulphur centers to ubiquinone.

- [5] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

Figure 16. Malate-Aspartate shuttle. (Harpers, p. 130)

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.

Figure 18. Transporter systems in the inner mitochondrial membrane.

(Harpers, p. 128)

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

Figure 17. Glycerophosphate shuttle. (Harpers, p. 130)

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

Figure 19. The Creatinine Phosphate shuttle. (Harpers, p. 131)

i.

- [6] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano

[BIOCHEMISTRY] 2.5 Electron Transport Chain and Oxidative Phosphorylation Balcueva, MD

VII. CLINICAL CORRELATIONS
1. Fatal infantile mitochondrial myopathy and renal dysfunction is the severe deficiency of the oxidoreductase enzymes in the
ETC.
2. MELAS is the deficiency of NADH-Q or cytochrome oxidase.
An inherited condition due to NADH-Q oxidoreductase (Complex I) or cytochrome oxidase (Complex IV) deficiency.
It is caused by a mutation in mitochondrial DNA and may be involved in Alzheimer's disease and diabetes mellitus.
3. Brown adipose tissue is a cold-induced thermogenesis due to production of uncoupling protein (UCP-1) that carries protons from
the mitochondrial matrix and uncouples ATP synthesis from respiration.
4. Leber is a hereditary optic neuropathy with sudden onset blindness from optic nerve death and results from single base mutation in
genes encoding complex I.
VIII. SUMMARY
Most of the energy released during the oxidation of different food trapped as reducing equivalents that are oxidized in the respiratory
chain.
Oxidation of reducing equivalents is tightly coupled to ATP production through ATP synthase.
Protons or electrons are passed on through a series of reductants and oxidants in the ETC to oxygen in complex IV to form water.
Oxidation through the different complexes also servs as a proton pump that translocates protons into the inter membrane space
producing a Chemiosmotic gradient for ATP synthesis.
IX. COMPETENCIES
1. Given a normal person, identify biochemical pathways or processes involved in ATP production in the mitochondria.
2. Apply the biochemical concepts and principles that will help explain the development of diseases associated with defects in the
electron transport system and oxidative phosphorylation.
3. Correlate the biochemical or molecular basis with the clinical manifestation of the disease, the findings on physical examination of the
patient and laboratory results.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

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

- [7] 14 - Estacion, Estillore, Estrada, Eugenio, Fabiania, Fandio, Favila, Fegarido, Feliciano