1
Respiratory chain
-Electron transport chain
The electron transport chain consists of a spatially separated series of redox
reactions in which electrons are transferred from a donor molecule to an acceptor
molecule. The underlying force driving these reactions is the Gibbs free energy of
the reactants and products. The Gibbs free energy is the energy available ("free")
to do work. Any reaction that decreases the overall Gibbs free energy of a system
is thermodynamically spontaneous.
The function of the electron transport chain is to produce a transmembrane proton
electrochemical gradient as a result of the redox reactions.[1] If protons flow back
through the membrane, they enable mechanical work, such as rotating bacterial
flagella. ATP synthase, an enzyme highly conserved among all domains of life,
converts this mechanical work into chemical energy by producing ATP,[2] which
powers most cellular reactions. A small amount of ATP is available from substratelevel
phosphorylation, for example, in glycolysis. In most organisms the majority of
ATP is generated in electron transport chains, while only some obtain ATP by
fermentation
Respiratory chain_Biochemistry-
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1
Transformation of energy in human
body
Chemical E of nutrient = work + heat
E of nutrient = BM + physical activity + reserves + heat
BM = basal methabolism
reserves = adipose tissue,
glycogene
Income of E Cost of E
Every work needs- ATP
chemical: synthesis of proteins.. ...
osmotical: transport of ions ...
mechanical: muscle contraction ...
Respiratory chain_Biochemistry-
10
2
Chemical E
of nutrient
heat
NADH+H+
FADH2
proton
gradient
heat heat
ATP
1 2 3
1 ....... metabolically dehydrogenation
2 ....... RCH = oxidation of reduced cofactors and reduction of O2 to
H2O
3 ....... Aerobic phosphorylation
............. High energetic system
Transformation of E – production of heat
Respiratory chain_Biochemistry-
10
3
nutrient E (kJ/g) Thermogenesis Source of E/day
Lipids 38 4 %  30 %
SAFA 5 %, MUFA 20 %, PUFA 5 %
CH + sugars 17 6 % 55 - 60 %
Proteins 17 30 % 10 - 15 %
Nutrients and E
Respiratory chain_Biochemistry-
10
4
5
Nutrients
O
OH
OH
OH
OH
CH2OH
I
0
0
0
0
-I
H3C
COOH
-II
-III
III
H3C CH
NH2
COOH
-III III0
Event. ox.n. C = 0,0
Event. ox.n. C = 0,0
Event. ox.n. C = -1,8 best C
Respiratory chain_Biochemistry-
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5
ATP
• Adenosine-triphosphate (ATP) is a nucleotide
triphosphate used in cells as a coenzyme. It is often
called the "molecular unit of currency" of intracellular
energy transfer.[1] ATP transports chemical energy within
cells for metabolism. It is one of the end products of
photophosphorylation, cellular respiration, and
fermentation and used by enzymes and structural
proteins in many cellular processes, including
biosynthetic reactions, motility, and cell division.[2] One
molecule of ATP contains three phosphate groups, and it
is produced by a wide variety of enzymes, including ATP
synthase, from adenosine diphosphate (ADP) or
adenosine monophosphate (AMP) and various
phosphate group donors. Substrate level
phosphorylation, oxidative phosphorylation in cellular
respiration, and photophosphorylation in photosynthesis
are three major mechanisms of ATP biosynthesis.Respiratory chain_Biochemistry-
10
6
7
Formation of ATP in body
Substrate phosphorylation
Aerobic phosphorylation
5%
95%
ATP
Respiratory chain_Biochemistry-
10
7
Formation and utilization of ATP
Respiratory chain_Biochemistry-
10
8
Aerobic phosphorylation (95 %)
ADP + Pi + energy H+-gradient  ATP
Substrate phosphorylation (5 %)
macroergic ~P + ADP  ATP + second product
!! Different: common phosphorylation
X-OH + ATP  X-O-P + ADP
Two ways of ATP formation
Respiratory chain_Biochemistry-
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9
10
Substrate phosphorylation
• ATP is produced after
conversion of macroergic
intermediates in metabolism of
nutrients
• succinyl-CoA (CC)
• 1,3-bisphosphoglycerate
(glycolysis)
• phosphoenolpyruvate
(glycolysis)
Aerobic
phosphorylation
• Connected to RCh
• For ATP synthesis is
used proton motive
force
Two ways of ATP formation
Respiratory chain_Biochemistry-
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10
CC Succinate formation: step5
Enzyme: succinyl CoA synthetase
GTP produced
GTP + ADP  GDP + ATP (NPTase)
Respiratory chain_Biochemistry-
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11
12
Phosphorylation of ADP by 1,3-
bisphosphoglycerate
N
N
N
N
NH2
O
OH OH
OPO
O
O
P
O
O
OPO
O
O
N
N
N
N
NH2
O
OH OH
OPO
O
O
P
O
O
O
ADP3-
C
C
OO
CH2
H OH
O
P
O
O
O
C
C
OO
CH2
H OH
O
P
O
O
O
P
O
O
O
1,3-bisphosphoglycerate
phosphoglycerate
ATP4Respiratory
chain_Biochemistry-
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12
13
Phosphorylation of ADP by
phosphoenolpyruvate
C
C
H H
OOOC P
O
O
O
+ ADP3-
H
+ C
C
H H
OHOOC
C
CH3
OOOC
enolpyruvate
pyruvate
phosphoenolpyruvate
+ ATP4Respiratory
chain_Biochemistry-
10
13
Mitochondrion
Double membrane
Outer membrane is
permeable
Inner membrane has
invaginations called
cristae
The electron
transport chain is
located in the inner
membrane
Respiratory chain_Biochemistry-
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Diagram of a mitochondrion
• Inner membrane
• Impermeable to most
• small molecules and
ions,
• including H+
• Contains:
• • Respiratory electron
• carriers (Complexes I–
IV)
• • ADP-ATP translocase
• • ATP synthase (FoF1)
• • Other membrane
• Transporters
• 80% of proteins
• Phospholipids
(kardiolipin)Respiratory chain_Biochemistry-
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15
Outer membrane
Freely permeable to
small molecules and ions
Respiratory chain_Biochemistry-
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16
17
Aerobic phosphorylation
Nutrient
(redused form of C)
CO2 + reduced cofactors
(NADH+H+, FADH2)
Reoxidation in
RCh
dehydrogenation
O2
Accumulation of E + H2O
ADP + Pi  ATP
OXIDATIVE
PHOSPHORYLATION
Respiratory chain_Biochemistry-
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17
18
Nutrients
O
OH
OH
OH
OH
CH2OH
I
0
0
0
0
-I
H3C
COOH
-II
-III
III
H3C CH
NH2
COOH
-III III0
Event. ox.n. C = 0,0
Event. ox.n. C = 0,0
Event. ox.n. C = -1,8 best C
Respiratory chain_Biochemistry-
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Formation of NADH in MM
• TCA
• isocitrate
2-oxoglutarate
malate
• -oxidation of FA
-hydroxyacyl-CoA
• Oxidation decarboxylation
pyruvate
2-oxoglutarate
• 2-oxo acids from Val, Leu, Ile
• Dehydrogenation of Ketone
bodies
• -hydroxybutyrate
• Dehydrogenation deaminatione
glutamate
Respiratory chain_Biochemistry-
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19
• Glycolysis
(dehydrogenation of glyceraldehyde-3-P)
• Gluconeogenesis
(dehydrogenation of lactate to pyruvate)
• Dehydrogenation of ethanol
(to acetaldehyde)
Formation of NADH in cytoplasm
Respiratory chain_Biochemistry-
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Formation of FADH2 in MM
• -Oxidation of FA
(dehydrogenation of alcyl-CoA)
• TCA
(dehydrogenation of succinate)
Respiratory chain_Biochemistry-
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21
22
Transport of NADH from cytoplasm to
matrix
• NADH from cytoplasm to matrix
• Inpermeable inner MM
• Change of H+ protones
• 2 shuttle mechanism
• aspartate/malate (heart, liver,kidney)
• glycerolphosphate (brain, muscle, kidney)
Respiratory chain_Biochemistry-
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22
23
Aspartate/malate shuttle
malát NAD
+
oxalacetát
Asp
AST
Asp
oxalacetát
transamination
NAD
+
MD hydrogenation
NADH H
+
+
NADH H
+
+
cytoplasm
matrixvnitřní
mitochondriální
membrána
dehydrogenation MD
AST
glutamate
2-oxoglutarate
glutamate
2-oxoglutarate
Antiporte with
2-oxoglutrate
Antiport
with
glutamate
matale
oxalacetate
matale
matale
oxalacetate
Inner mitoch.
membraneRespiratory chain_Biochemistry-
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23
Glycerolphosphate
shuttle
Mozek sval
GPD
CH2
C
CH2
O
O
OH
P
dihydroxyacetonP
NAD
NADH + H
CH2
C
CH2 O
OH
H OH
P
glycerol-3-Pt
Q
QH2
ubichinon
ubichinol
inner
mitochondrial
membrane
glycerolphosphatedehydrogenase
GPD(NAD+) GPD(FAD+)
Brain, muscle
Respiratory chain_Biochemistry-
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Respiratory chain
• An electron transport chain (ETC) couples
electron transfer between an electron donor
(such as NADH) and an electron acceptor (such
as O2) with the transfer of H+ ions (protons)
across a membrane. The resulting
electrochemical proton gradient is used to
generate chemical energy in the form of
adenosine triphosphate (ATP). Electron
transport chains are the cellular mechanisms
used for extracting energy from sunlight in
photosynthesis and also from redox reactions,
such as the oxidation of sugars (respiration).
Respiratory chain_Biochemistry-
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The Components of the Electron
Transport Chain
• The electron transport chain of the
mitochondria is the means by which
electrons are removed from the reduced
carrier NADH and transferred to oxygen to
yield H2O
Respiratory chain_Biochemistry-
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27
Respiratory chain
+ H+
Nutrients
dehydrogenation
matrix
Inner mitochondrial membrane
Intermembrane space
coenzyme Q cytochroms
O2 ATP
2 e 2 e 2 e
n H
NADH FADH2
H2O
n Hn H n H
Respiratory chain_Biochemistry-
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27
28
Inner mitochondrial membrane
• Large surface
• High concetration of proteins (enzymes, shuttles)
• Permeable for small no charge moleculs
• Non permeable for ions, organic substrates
• The mitochondrial inner membrane forms internal
compartments known as cristae, which allow greater space for
the proteins such as cytochromes to function properly and
efficiently. The electron transport chain is located on the inner
membrane of the mitochondria. Within the inner mitochondrial
membrane are also transport proteins that transport in a highly
controlled manner metabolites across this membrane.
Respiratory chain_Biochemistry-
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28
Respiratory chain
•Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the
Krebs cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes
them to coenzyme Q (ubiquinone; labeled UQ), which also receives electrons from
complex II (succinate dehydrogenase; labeled II). UQ passes electrons to
complex III (cytochrome bc1 complex; labeled III), which passes them to
cytochrome c (cyt c). Cyt c passes electrons to Complex IV (cytochrome c
oxidase; labeled IV), which uses the electrons and hydrogen ions to reduce
molecular oxygen to water.
Respiratory chain_Biochemistry-
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29
30
Reduced coffactors
I.
CC
-oxidation
Q
I.
II.
shuttel
NADH + H
NAD
+
matrix
cytoplasm
FAD
FAD FAD
glycerol-P DHAP
succinate
fumarate
alkanoyl-CoA
alkenoyl-CoA
Respiratory chain_Biochemistry-
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30
• Energy obtained through the transfer of electrons (black arrows)
down the ETC is used to pump protons (red arrows) from the
mitochondrial matrix into the intermembrane space, creating an
electrochemical proton gradient across the mitochondrial inner
membrane (IMM) called ΔΨ. This electrochemical proton gradient
allows ATP synthase (ATP-ase) to use the flow of H+ through the
enzyme back into the matrix to generate ATP from adenosine
diphosphate (ADP) and inorganic phosphate. Complex I (NADH
coenzyme Q reductase; labeled I) accepts electrons from the Krebs
cycle electron carrier nicotinamide adenine dinucleotide (NADH),
and passes them to coenzyme Q (ubiquinone; labeled UQ), which
also receives electrons from complex II (succinate dehydrogenase;
labeled II). UQ passes electrons to complex III (cytochrome bc1
complex; labeled III), which passes them to cytochrome c (cyt c).
Cyt c passes electrons to Complex IV (cytochrome c oxidase;
labeled IV), which uses the electrons and hydrogen ions to reduce
molecular oxygen to water.
• Four membrane-bound complexes have been identified in
mitochondria. Each is an extremely complex transmembrane
structure that is embedded in the inner membrane. Three of them
are proton pumps. The structures are electrically connected by lipidsoluble
electron carriers and water-soluble electron carriers. The
overall electron transport chain:
Respiratory chain_Biochemistry-
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31
Sequence of electron carriers in the respiratory chain
Complex I
proton pump
Complex II, does not
pump protons
Complex III
proton pump
Complex IV
proton pump
Coenzyme Q
electron shuttle
Cytochrome c
electron shuttle
Ch
Respiratory chain_Biochemistry-
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32
Summary of
electron-transport
chain
Respiratory chain_Biochemistry-
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33
34
Enzyme complexes of RCh
complexes name cofactors Transfer e-
I.
II.
III.
IV.
NADH-dehydrogenase
succinatedehydrogenase
cytochrom-c-reductase
cytochrom-c-oxidase
FMN, Fe-S
FAD, Fe-S, cyt b
Fe-S, cyt b, c1
cyt a, a3, Cu
NADH  Q
FADH2  Q
Q  cyt c
cyt c  O2
Respiratory chain_Biochemistry-
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35
Redox pairs in RCh
Oxid / Red form E´(V)
NAD+ / NADH+H+
FAD / FADH2
Ubichinon (Q) / Ubichinol (QH2)
Cytochrom c1 (Fe3+ / Fe2+)
Cytochrom c (Fe3+ / Fe2+)
Cytochrom a3 (Fe3+ / Fe2+)
O2 / 2H2O
-0,32
0,00
0,10
0,22
0,24
0,39
0,82
Respiratory chain_Biochemistry-
10
35
• Complex I
• In Complex I (NADH dehydrogenase, also called NADH:ubiquinone
oxidoreductase; EC 1.6.5.3) two electrons are removed from NADH and
transferred to a lipid-soluble carrier, ubiquinone (Q). The reduced product,
ubiquinol (QH2) freely diffuses within the membrane, and Complex I
translocates four protons (H+) across the membrane, thus producing a
proton gradient. Complex I is one of the main sites at which premature
electron leakage to oxygen occurs, thus being one of the main sites of
production of superoxide.[3]
• The pathway of electrons occurs as follows:
• NADH is oxidized to NAD+, by reducing Flavin mononucleotide to FMNH2
in one two-electron step. FMNH2 is then oxidized in two one-electron steps,
through a semiquinone intermediate. Each electron thus transfers from the
FMNH2 to an Fe-S cluster, from the Fe-S cluster to ubiquinone (Q). Transfer
of the first electron results in the free-radical (semiquinone) form of Q, and
transfer of the second electron reduces the semiquinone form to the
ubiquinol form, QH2. During this process, four protons are translocated from
the mitochondrial matrix to the intermembrane space. [3]
Respiratory chain_Biochemistry-
10
36
NADH-Q
oxidoreductas
e
Respiratory chain_Biochemistry-
10
37
Structure of NADH-Q oxidoreductase
EM at moderate resolution
Matrix
side
Consists of at least
34 polypeptide chains
NADH is oxidized in the arm,
& electrons are transferred to
reduce Q in the membrane
Respiratory chain_Biochemistry-
10
38
Oxidation states of quinones
Coenzyme Q (Q) is a quinone derivative
with a long isoprenoid tail,
also known as ubiquinone
Reduction proceeds through
a semiquinone anion intermiediate (Q.-)
Respiratory chain_Biochemistry-
10
39
Oxidation states of flavins
Respiratory chain_Biochemistry-
10
40
Iron-sulfur clusters Undergo oxidation-reduction reactio
Single Fe ion
+ 4 Cys cluster
2Fe-2S cluster,
bridge of sulfide ions 4Fe-4S cluster
Respiratory chain_Biochemistry-
10
41
Coupled electron-proton transfer reactions
Reduction of Q can result in the uptake of 2 protons
Respiratory chain_Biochemistry-
10
42
Respiratory chain_Biochemistry-
10
43
44
NADH+H+ + FMN  NAD+ + FMNH2
Fe-S
FMN + 2 H+ + 2 e-
NADH-dehydrogenase
Respiratory chain_Biochemistry-
10
44
45
complex I - first „proton pump“ to
intermembrane space
VMM
intermembrane space
matrix
Q
2 H
QH2
NADH + H
NAD
FMN
2 H
2 H
2 e
FeS
(3 - 4 H )?
IMM
Respiratory chain_Biochemistry-
10
45
Complex II
• In Complex II (succinate dehydrogenase;
EC 1.3.5.1) additional electrons are
delivered into the quinone pool (Q)
originating from succinate and transferred
(via FAD) to Q. Complex II consists of four
protein subunits: SDHA, SDHB, SDHC,
and SDHD. Other electron donors (e.g.,
fatty acids an glycerol 3-phosphate) also
direct electrons into Q (via FAD).
Respiratory chain_Biochemistry-
10
46
Succinate-Q
reductase
Respiratory chain_Biochemistry-
10
47
48
Complex II - FADH2
matrix
VMM Q
2 H
QH2
2 e
succinate
fumarte
FAD
FeS
CC
cyt b
IMM
Respiratory chain_Biochemistry-
10
48
49
Ubichinon –mobile cofactor
O
O
O
O
• vysoce lipofilní, polyisoprenoidní řetězec je zakotven v VMM
• benzochinonový kruh se může pohybovat od jednoho kraje
membrány k druhému, sbírat red. ekvivalenty a odevzdávat je na
cytochromy
Respiratory chain_Biochemistry-
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49
Complex III
• In Complex III (cytochrome bc1 complex; EC 1.10.2.2), the Q-cycle
contributes to the proton gradient by an asymmetric
absorption/release of protons. Two electrons are removed from QH2
at the QO site and sequentially transferred to two molecules of
cytochrome c, a water-soluble electron carrier located within the
intermembrane space. The two other electrons sequentially pass
across the protein to the Qi site where the quinone part of
ubiquinone is reduced to quinol. A proton gradient is formed by two
quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e)
at the Qi site. (in total six protons are translocated: two protons
reduce quinone to quinol and four protons are released from two
ubiquinol molecules).
• When electron transfer is reduced (by a high membrane potential or
respiratory inhibitors such as antimycin A), Complex III may leak
electrons to molecular oxygen, resulting in superoxide formation.
Respiratory chain_Biochemistry-
10
50
51
Complex III a Q-cyclus transfer 2  2H+
matrix
IMM
Intermembrane space
2 H+ 2 H+
OH
OH
O
O
cyt c
FeS
cyt c1
cyt b
2 e
2 e
cyt c
Q-cyclus
2 x 2 H
Respiratory chain_Biochemistry-
10
51
III- Q- Cytochrome c oxidoreductase
Respiratory chain_Biochemistry-
10
52
Q-cytochrome c oxidoreductase
Homodimer with 11
distinct polypeptides
Major prosthetic guoups:
3 hemes, & a 2Fe-2S
cluster,
they mediate electrontransfer
between
quinones in the
membrane &
cytochrome c in the
intermembrane space
Respiratory chain_Biochemistry-
10
53
Attachment of heme group in c-type cytochromes
A cytochrome is an electron-transferring
protein that contains a heme
prosthetic group
Respiratory chain_Biochemistry-
10
54
Complex IV
• In Complex IV (cytochrome c oxidase; EC
1.9.3.1), sometimes called cytochrome A3, four
electrons are removed from four molecules of
cytochrome c and transferred to molecular
oxygen (O2), producing two molecules of water.
At the same time, four protons are removed from
the mitochondrial matrix (although only two are
translocated across the membrane), contributing
to the proton gradient. The activity of
cytochrome c oxidase is inhibited by cyanide.
Respiratory chain_Biochemistry-
10
55
56
Complex IV - second H+ pump
IMM
?
cyt a Cu
2 e
cyt c
2 H
2 e
cyt a3
1/2 O2
O2- H2O
2 H
matrix
Intermembrane spaceRespiratory chain_Biochemistry-
10
56
57
Proton motive force :2 parts
inout Electric part
= differences of membrane potentials
Concentration part = differences in pH inout pHpHpH 
Using of proton motive force
• Synthesis ofATP
•Heat
• Active transports of metabolits -IMM
Respiratory chain_Biochemistry-
10
57
Proton motive force
Respiratory chain_Biochemistry-
10
58
The enzyme complex consists of an F0
subcomplex which is a disk of “C” protein
subunits. Attached is a γ-subunit in the form
of a “bent axle.”
Protons passing through the disk of “C” units
cause it and the attached γ-subunit to rotate.
The γ-subunit fits inside the F1 subcomplex of
three α- and three β-subunits, which are fixed
to the membrane and do not rotate.
ADP and Pi are taken up sequentially by the
β-subunits to form ATP, which is expelled as
the rotating γ-subunit squeezes each βsubunit
in turn.
Thus, three ATP molecules are generated per
revolution. For clarity, not all the subunits that
have been identified are shown—eg, the
“axle” also contains an ε-subunit.
Mechanism of ATP production by ATP synthase.
Respiratory chain_Biochemistry-
10
59
60
Synthesis of ATP
H+
H+
H+
H+
H+
H+
H+
H+
H+
Fo
OSCP
F1
ADP + Pi ATP
Respiratory chain_Biochemistry-
10
60
61
Synthesis of ATP
• ATP-syntaase -3 parts
• Fo channel for H+
• F1 in matrix, synthesis of ATP
• OSCP (oligomycin sensitivity
conferring protein)
Respiratory chain_Biochemistry-
10
61
62
Connection of RCh and OPh
• RCh and OPh – non permeable IMM for H+
• Only way for H+ to matrix- Fo ATP-synthase
Respiratory chain_Biochemistry-
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62
Uncouplers and Inhibitors
Specific inhibitors were used to distinguish the electron transport system from the
phosphorylation system and helped to define the sequence of redox carriers along the
respiratory chain. If the chain is blocked then all the intermediates on the substrate side of
the block become more reduced, while all those on the oxygen side become more oxidized.
It is easy to see what has happened because the oxidized and reduced carriers often differ
in their spectral properties. If a variety of different inhibitors are available then many of the
respiratory carriers can be placed in the correct order.Respiratory chain_Biochemistry-
10
63
Respiratory chain_Biochemistry-
10
64
There are six distinct types of
poison which may affect
mitochondrial function:
• 1) Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone & TTFA)
block respiration in the presence of either ADP or uncouplers.
• 2) Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen
consumption after adding ADP, but have no effect on uncoupler-stimulated
respiration.
• 3) Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the obligatory
linkage between the respiratory chain and the phosphorylation system which is
observed with intact mitochondria.
• 4) Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either
prevent the export of ATP, or the import of raw materials across the the
mitochondrial inner membrane.
• 5) Ionophores (e.g. valinomycin, nigericin) make the inner membrane
permeable to compounds which are ordinarily unable to cross.
• 6) Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or
more of the TCA cycle enzymes, or an ancillary reation.
• Some of the best-known compounds are listed below:
Respiratory chain_Biochemistry-
10
65
66
Uncouples
• abolish the obligatory linkage between the respiratory
chain and the phosphorylation system
• abolish proton gradient without gain of ATP
• Creation of heat
• RCh is running
• OPh is closed
• Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP)
abolish the obligatory linkage between the respiratory
chain and the phosphorylation system which is observed
with intact mitochondria.
Respiratory chain_Biochemistry-
10
66
67
Uncouples
n H+
DŘ
n H
+
n H
+
n H
+
n H
+
ATP
heat
X
uncouple
ATP synthase
Respiratory chain_Biochemistry-
10
67
68
2,4-Dinitrophenol
OH
NO2
O2N
Respiratory chain_Biochemistry-
10
68
69
Thermogenin-Biological Uncoupling
• Protein with channel for H+, adipose tissue,
• Brown adipose tissue, new born child,
• Uncoupling the ETS from oxidative phosphorylation speeds
metabolism, and generates heat. Some mammals lacking fur use this
function in brown adipose tissue as a way of generating heat.
• One such process is called nonshivering thermogenesis that occurs
in cells in the neck and upper back. The mitochondria of brown
adipose tissue cells contain a protein called thermogenin (or
uncoupling protein - UCP). Thermogenin acts as a channel to
permeabilize these cells' inner mitochondrial membrane to protons.
Normally, ADP, ATP, GDP, and GTP are present in high enough
concentrations to block the flow of protons through it. However,
thermogenin in the mitochondria of these cells is activated to
uncoupling by the presence of free fatty acids. Free fatty acids can be
generated in these cells by the hormone norepinephrine, which
through second messengers (including cAMP) activates hormonesensitive
triacylglycerol lipase to cleave fats to release fatty acids.
Thus, brown adipose tissue cells respond to norepinephrine by
uncoupling the ETS from oxidative phosphorylation, speeding
metabolism and generating heat, at the expense of metabolic energy.Respiratory chain_Biochemistry-
10
69
Proton gradient is interconvertible form of free energy
Respiratory chain_Biochemistry-
10
70
71
Transport of metabolits over IMM
• Non permeable – shuttle syntems
• Source of E- proton motive force of RCh
• Secondary active transport
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• O2, H2O, NH3 - free
• FA - carnitin
• pyruvare - symport with H+
• CC, AA acids - specific transporters
• hydrogenphosphate – exchange for OH•
malate- exchange for 2-oxoglutarate (shuttle)
• aspartate – exchange for glutamate (shuttle)
• ATP – exchange for ADP
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Mitochondrial transporters
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74
matrix
OH-
H2PO4
pyruvate
H+
malate
malate
citrát + H+
ATP
ADP
cytosol
HPO4
2Respiratory
chain_Biochemistry-
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Respiratory chain_Biochemistry-
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Glycerophosphate shuttle for transfer of
reducing equivalents from the cytosol into the
mitochondrion.
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Malate shuttle for transfer of reducing equivalents from
the cytosol into the mitochondrion. 1 Ketoglutarate transporter;
2 , glutamate/aspartate transporter (note the proton symport with
glutamate).
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The creatine phosphate shuttle of
heart and skeletal muscle. The shuttle
allows rapid transport of high-energy
phosphate from the mitochondrial
matrix into the cytosol.
CKa, creatine kinase
concerned with large requirements for
ATP, eg, muscular contraction; CKc,
creatine kinase for maintaining
equilibrium between creatine and
creatine phosphate and ATP/ADP;
CKg, creatine kinase coupling glycolysis
to creatine phosphate synthesis; CKm,
mitochondrial creatine kinase mediating
creatine phosphate production
from ATP formed in oxidative
phosphorylation; P, pore protein in outer
mitochondrial membrane.
The creatine phosphate shuttle
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79
Inhibitors
RCh
• rotenon, barbital (I)
• malonate (II)
• antimycin A (III)
• dimerkaprol (III)
• CO, CN-, SH-, N3
- (IV)
ATP-synthase
• oligomycin
ATP/ADP-translocase
Bongcrecid acid
• atractylosid
There are several well-known drugs and toxins that inhibit oxidative phosphorylation.
Although any one of these toxins inhibits only one enzyme in the electron transport
chain, inhibition of any step in this process will halt the rest of the process. For
example, if oligomycin inhibits ATP synthase, protons cannot pass back into the
mitochondrion.[84] As a result, the proton pumps are unable to operate, as the
gradient becomes too strong for them to overcome. NADH is then no longer oxidized
and the citric acid cycle ceases to operate because the concentration of NAD+ falls
below the concentration that these enzymes can use.
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There are six distinct types of
poison which may affect
mitochondrial function:
• 1) Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone & TTFA)
block respiration in the presence of either ADP or uncouplers.
• 2) Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen
consumption after adding ADP, but have no effect on uncoupler-stimulated
respiration.
• 3) Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the obligatory
linkage between the respiratory chain and the phosphorylation system which is
observed with intact mitochondria.
• 4) Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either
prevent the export of ATP, or the import of raw materials across the the
mitochondrial inner membrane.
• 5) Ionophores (e.g. valinomycin, nigericin) make the inner membrane
permeable to compounds which are ordinarily unable to cross.
• 6) Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or
more of the TCA cycle enzymes, or an ancillary reation.
• Some of the best-known compounds are listed below:
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Cyanide poisoning
• Many cyanides are highly toxic. The cyanide anion is an inhibitor of the
enzyme cytochrome c oxidase (also known as aa3) in the fourth complex of
the electron transport chain (found in the membrane of the mitochondria of
eukaryotic cells). It attaches to the iron within this protein. The binding of
cyanide to this cytochrome prevents transport of electrons from cytochrome
c oxidase to oxygen. As a result, the electron transport chain is disrupted,
meaning that the cell can no longer aerobically produce ATP for energy.[18]
Tissues that depend highly on aerobic respiration, such as the central
nervous system and the heart, are particularly affected. This is an example
of histotoxic hypoxia.[19]
• The most hazardous compound is hydrogen cyanide, which is a gas at
ambient temperatures and pressure and can therefore be inhaled. For this
reason, an air respirator supplied by an external oxygen source must be
worn when working with hydrogen cyanide. Hydrogen cyanide is produced
when a solution containing a labile cyanide is made acidic, because HCN is
a weak acid. Alkaline solutions are safer to use because they do not evolve
hydrogen cyanide gas. Hydrogen cyanide may be produced in the
combustion of polyurethanes; for this reason, polyurethanes are not
recommended for use in domestic and aircraft furniture. Oral ingestion of a
small quantity of solid cyanide or a cyanide solution as little as 200 mg, or to
airborne cyanide of 270 ppm is sufficient to cause death within minutes.[19]
• Organic nitriles do not readily release cyanide ions, and so have low
toxicities. By contrast, compounds such as trimethylsilyl cyanide
(CH3)3SiCN readily release HCN or the cyanide ion upon contact with
water.[citation needed] Respiratory chain_Biochemistry-
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• Antidote
• Hydroxocobalamin reacts with cyanide to form cyanocobalamin,
which can be safely eliminated by the kidneys. This method has the
advantage of avoiding the formation of methemoglobin (see below).
This antidote kit is sold under the brand name Cyanokit and was
approved by the FDA in 2006.[20]
• An older cyanide antidote kit included administration of three
substances: amyl nitrite pearls (administered by inhalation), sodium
nitrite, and sodium thiosulfate (administered by infusion). The goal of
the antidote was to generate a large pool of ferric iron (Fe3+) to
compete with cyanide cytochrome a3 (so that cyanide will bind to
the antidote rather that the enzyme). The nitrites oxidize hemoglobin
to methemoglobin, which competes with cytochrome oxidase for the
cyanide ion. Cyanmethemoglobin is formed and the cytochrome
oxidase enzyme is restored. The major mechanism to remove the
cyanide from the body is by enzymatic conversion to thiocyanate by
the mitochondrial enzyme rhodanese. Thiocyanate is a relatively
non-toxic molecule and is excreted by the kidneys. To accelerate
this detoxification, sodium thiosulfate is administered to provide a
sulfur donor for rhodanese, needed in order to produce thiocyanate.
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Respiratory chain_Biochemistry-
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84
ROS, reactive oxygen
species
Reactive oxygen species (ROS) are chemically reactive molecules containing
oxygen. Examples include oxygen ions and peroxides. ROS form as a natural
byproduct of the normal metabolism of oxygen and have important roles in cell
signaling and homeostasis.[1] However, during times of environmental stress (e.g.,
UV or heat exposure), ROS levels can increase dramatically.[1] This may result in
significant damage to cell structures. Cumulatively, this is known as oxidative
stress. ROS are also generated by exogenous sources such as ionizing radiation.
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Reactive Oxygen Species (ROS)
Radicals:
O2
.- Superoxide
OH. Hydroxyl
RO2
. Peroxyl
RO. Alkoxyl
HO2
. Hydroperoxyl
Non-Radicals:
H2O2 Hydrogen peroxide
HOCl- Hypochlorous acid
O3 Ozone
1O2 Singlet oxygen
ONOO- Peroxynitrite
Reactive Nitrogen Species (RNS)
Radicals:
NO. Nitric Oxide
NO2
. Nitrogen dioxide
Non-Radicals:
ONOO- Peroxynitrite
ROONO Alkyl peroxynitrites
N2O3 Dinitrogen trioxide
N2O4 Dinitrogen tetroxide
HNO2 Nitrous acid
NO2
+ Nitronium anion
NO- Nitroxyl anion
NO+ Nitrosyl cation
NO2Cl Nitryl chlorideRespiratory chain_Biochemistry-
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“Longevity” of reactive species
Reactive Species Half-life
Hydrogen peroxide
Organic hydroperoxides ~ minutes
Hypohalous acids
Peroxyl radicals ~ seconds
Nitric oxide
Peroxynitrite ~ milliseconds
Superoxide anion
Singlet oxygen ~ microsecond
Alcoxyl radicals
Hydroxyl radical ~ nanosecond
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“An imbalance favoring prooxidants and/or disfavoring
antioxidants, potentially leading to damage” -H. Sies
Antioxidants
Prooxidants
Oxidative Stress
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Radical-mediated reactions
Addition
R. + H2C=CH2 R-CH2-CH2
.
Hydrogen abstraction
R. + LH RH + L.
Electron abstraction
R. + ArNH2 R- + ArNH2
.+
Termination
R. + Y. R-Y
Disproportionation
CH3CH2
. + CH3CH2
. CH3CH3 + CH2=CH2
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Hydroxyl radical (.OH)
O2
.- + Fe3+ O2 + Fe2+ (ferrous)
H2O2 + Fe2+ OH- + .OH + Fe3+ (ferric)
O2
.- + H2O2 OH- + O2 + .OHHaber-Weiss
Fenton
•Transition metal catalyzed
•Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones)
•Fe2+ is an extremely reactive oxidant
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Important Enzyme-Catalyzed Reactions
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Biological Pathways for Oxygen Reduction
From:McMurryandCastellion“Fundamentalsofgeneral,organicandbiologicalchemistry”
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Endogenous sources of ROS and RNS
Mitochondria
Lysosomes
Peroxisomes
Endoplasmic Reticulum
Cytoplasm
Microsomal Oxidation,
Flavoproteins,
CYP enzymes
Myeloperoxidase
(phagocytes)
Electron transport
Oxidases,
Flavoproteins
Plasma Membrane
Lipoxygenases,
Prostaglandin synthase
NADPH oxidase
Xanthine Oxidase,
NOS isoforms
Fe
Cu
Transition
metals
II. Sources of ROS
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93
Antioxidant systems of the organism
1. Enzymes (endogenous), superoxide dismutase , catalase , glutathione
peroxidase
2. Second high molecular weight antioxidants ( endogenous ), transferrin,
ferritin , ceruloplasmin al., Bind free metal ions
3. Third low molecular weight antioxidants ( exogenous , endogenous ),
-reducing substances with the phenolic -OH ( tocopherol , flavonoids, urate)
- reducing substances with enolic OH ( ascorbate )
- reducing substance having -SH group ( glutathione, dihydrolipoát )
- substances with an extensive system of conjugated double bonds ( carotenoids,
retinol, bilirubin)
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