Electron transport chain

@2017

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Electron transport chain

• All oxidative steps in the degradation of carbohydrates, fats, and amino

acids converge in the final stage of cellular respiration in which the energy
of oxidation drivers the synthesis of ATP – oxidative phosphorylation
• This accounts for most of the ATP generated
• Occurs in the mitochondria.
• Oxidative phosphorylation involves the reduction of O2 to H2O with electrons
donated by NADH and FADH2

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Biochemical anatomy of the mitochondria
Outer membrane.
Freely permeable to small
molecules and ions

Inner membrane.
Impermeable to most small
molecules and ions, including
H+. Contains:Respiratory
electron carriers (complexes
I-IV), ADP-ATP translocases,
ATP synthase and other
membrane transporters

Matrix. Contains: pyruvate

dehydrogenase complex, citric acid
cycle enzymes, fatty acid β–oxidation
enzymes, amino acid oxidation
enzymes, many other enzymes,
many soluble metabolic
intermediates, ATP, ADP, Pi, Mg2+,
Ca2+, K+, DNA etc

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• The selectively permeable inner mitochondrial membrane segregates the
intermediates and enzymes of the cytosolic metabolic pathways from those
processes occurring in the matrix
• Specific transporters carry pyruvate, fatty acids and amino acids or their α-keto
derivatives into the matrix

Electron-transfer reactions in the mitochondria

Electrons carriers
 Nicotinamide nucleotides
• NADH carries electrons from catabolic reactions to the point of entry into the
electron transport chain, NADH dehydrogenase complex

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 Flavoproteins
• Contains very tightly sometimes covalently bound flavin nucleotides, either FAD or
FMN
• The oxidized flavin nucleotide can accept either one electron (yielding the
semiquinone form ) or two (yielding FADH2 or FMNH2)
• Because flavoproteins can participate in one- or two- electron transfers they can
serve as intermediates in which two electrons are donated ( as in dehydrogenations)
or those in which only one electron is accepted ( as in reduction of quinone to a
hydroquinone)

 Ubiquinone (coenzyme Q)
• Its lipid soluble with along isoprenoid side chain

• Can accept one electron to become a semiquinone radical (QH.) or two to form
ubiquinol (QH2)

• Like flavoprotein carriers it can act at a junction between a two-electron donor and a
one–electron acceptor
• Because its small and hydrophobic, it is freely diffusible within the lipid bilayer of
the inner mitochondrial membrane and can shuttle reducing equivalents
between other less mobile carriers in the membrane
• And because it carries both electrons and protons, it plays a central role in 5
coupling electron flow to proton movements
 The cytochromes
• Are proteins with iron-containing heme prosthetic group

 Iron-sulfur proteins
• In these proteins the iron is present not as heme but in association with inorganic
sulfur atoms or with the sulfur atoms of cysteine residues or both
• These iron–sulfur centers (Fe-S) range from simple structures with a single Fe atom
to more complex Fe-S centers with two or four Fe atoms
• All iron–sulfur proteins participate in one-electron transfers in which one iron atom of
the iron–sulfur cluster is oxidized or reduced

Most of the electron carriers are integral proteins with prosthetic groups capable of
accepting or donating either one or two electrons
• Three types of electron transfers occur in oxidative phosphorylation
• Direct transfer as in the reduction of Fe3+ to Fe2+
• Transfer as a hydrogen atom (H+ + ē)
• Transfer as a hydride ion (:H–) 6
Electron carriers function in order of increasing reduction potential, because
electrons tend to flow from carriers of lower E′o to carriers of higher E′o

Standard reduction potentials

Redox reaction (half-reaction) E′o (V)
2H+ + 2ē → H2 –0.414
NAD+ + H+ + 2ē → NADH –0.320
NADP+ + H+ + 2ē → NADPH –0.324
NADH dehydrogenase (FMN) + 2H+ + 2ē →NADH dehydrogenase(FMNH2) –0.30
Ubiquinone + 2H+ + 2ē → Ubiquinol 0.045
Cyt b (Fe3+) + ē → Cyt b (Fe2+) 0.077
Cyt c1 (Fe3+) + ē → Cyt c1 (Fe2+) 0.22
Cyt c (Fe3+) + ē → Cyt c (Fe2+) 0.254
Cyt a (Fe3+) + ē → Cyt a (Fe2+) 0.29
Cyt a3 (Fe3+) + ē → Cyt a3 (Fe2+) 0.55
½ O2 + 2H+ + 2ē → H2O 0.816

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Electron carriers function in multienzyme complexes
• The reduction potential increases as electrons flow down the respiratory chain to O2
• As most cytochromes have higher redox potentials than iron-sulfur proteins they
generally serve as electron carriers near the O2 end of the chain, where as iron-sulfur
centers serve as carriers near the NADH end
4H+
NADH NAD+
V
–0.3 4(?2)H+ Complex II is not a
Complex proton pump because
I C II its free energy change
– 0.045 Q is too small
– 0.077
Complex 2(?4)H
+
III
0.254
Cyt C
0.29 The formular ΔG0= – nF ΔE’0
shows that free energy
Complex
IV change is proportional to ΔE’0

0.82

2H + 1/2O2 H2O
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Direction of electron flow
 Sequence of electron carriers in the respiratory chain.
(In brackets are the prosthetic groups of the respective enzymes)
Pyruvate
(Lipoate) Glutamate, malate, isocitrate,3-
FAD hydroxybutyrate, 3-hydroxyacyl-
α-Ketoglutarate NADH
CoA, proline

NADH dehydrogenase (NADH:ubiquinone oxidoreductase) (FMN, Fe-S)

(Complex I)
Glycerol- 3-phosphate
FAD Q Succinate-Q reductase (Succinate
Fatty acyl-CoA dehydrogenase )(Complex II)(FAD, Fe-S)

Cytochrome reductase (Ubiquinone:cytochrome c(Heme b, Heme c1, Fe-S)

oxidoreductase ) (Complex III)
Cyt c

Cytochrome oxidase (Complex IV) (Heme a, Heme a3, CuA, CuB)

O2 9
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 Complex I (NADH dehydrogenase) : Transfers electrons from NADH to
ubiquinone
• The reduced NADH of the chain is oxidized by a metalloflavoprotein
enzyme NADH dehydrogenase
• It’s a large, L-shaped enzyme composed of 42 polypeptide chains that
includes an FMN-containing flavoprotein and at least 6 iron-sulfur clusters

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NADH:ubiquinone oxidoreductase (Complex I).
• Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from
which two electrons pass through a series of Fe-S centers to the iron sulfur
protein N-2 in the matrix arm of the complex
• Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2
which diffuses into the lipid bilayer.
• This electron transfer also drives the expulsion from the matrix of four
protons per pair of electrons.
• Proton flux produces an electrochemical potential across the inner
mitochondrial membrane (N side negative, P side positive), which
conserves some of the energy released by the electron-transfer reactions.
This electrochemical potential drives ATP synthesis
• Amytal ( a barbiturate), rotenone (a plant product commonly used as an
insecticide) and piericidin (an antibiotic) inhibit electron flow from Fe-S
centers to ubiquinone
• Ubiquinol (QH2, the fully reduced form diffuses in the inner mitochondrial
membrane from complex I to complex III)

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 Complex II (Succinate-Q reductase). Transfers electrons from succinate to
ubiquinone
• Succinate-Q reductase has two prosthetic groups; FAD and Fe-S centers
• Electrons pass from succinate to FAD, then through the Fe-S centers to ubiquinone
• Other substrates for mitochondrial dehydrogenases pass their electrons into the
respiratory chain at the level of ubiquinone, but not through complex II

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• The first step in the β oxidation of fatty acyl-CoA, catalyzed by the
flavoprotein acyl-CoA dehydrogenase involves the transfer of electrons from
substrate to the FAD of the dehydrogenase, then to the electron transferring
flavoprotein (ETF), which in turn passes the electrons to ETF: ubiquinone
oxidoreductase
– The enzyme ETF:ubiquinone oxidoreductase passes the electrons into the
respiratory chain by reducing ubiquinone
• Glycerol 3-phosphate formed from glycerol released by triacylglycerol
breakdown and reduction of dihydroxyacetone phosphate is oxidized by
glycerol 3-phosphate dehydrogenase
– This enzyme is located on the outer face of the inner mitochondrial membrane
– It channels electrons into the respiratory chain by reducing ubiquinone
– The important role of glycerol 3-phosphate dehydrogenase is shuttling reducing
equivalents from cytosolic NADH into the mitochondrial matrix

• Succinate-Q reductase complex and other enzymes that transfer electrons

from FADH2 to O2 are not proton pumps because free energy changes of
the catalyzed reaction is too small. Result –less ATP formed from oxidation
of FADH than NADH

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 Complex III (Cytochrome reductase): transfers electrons from ubiquinol
to cytochrome c

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• Cytochrome reductase contains cytochromes b and c1 and iron–sulfur
protein
• Cyt b has two heme groups bL(b-566) and bH(b-560)
• Ubiquitol transfers one of its two electrons to the Fe-S cluster in the
enzyme. This electron is then shuttled sequentially to cytochrome c1 and c
• The one electron transfer converts ubiquinol(QH2) into the semiquinone(Q˙–)
• Q˙– rapidly transfers its electron to bL to form Q, which is free to diffuse into
the membrane
• Heme bL then reduces bH which in turn reduces the bound Q on the
cytosolic side to form Q˙– .
• A second molecule of QH2 then reacts with the complex as above. But this
time bH reduces the bound Q˙– rather than Q to complete the cycle
• The Q cycle accommodates the switch between the two-electron carrier
(QH2) to the one-electron carriers- cytochromes b, c1 and c
• Cytochrome c is a soluble protein of the intermembrane space. After
accepting an electron cytochrome c moves to complex IV to donate the
electron
• The flow of a pair of electrons through the complex leads to the effective net
transfer of 4(?2)H+ to the cytosolic side. Dimercaprol and Antimycin A
interfere with electron flow between cyt b and c1
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 Complex IV (Cytochrome oxidase): Cytochrome c to O2
• Carries electrons from cyt c to molecular oxygen
• Complex IV is a large enzyme (13 subunits) of the inner mitochondrial
membrane
• Three subunits are critical to the function
• Subunit II contains 2 copper ions complexed with the –SH groups of
cysteine (CuA)
• Subunit I contains two heme groups designated as a and a3, and
another copper ion (CuB)
• Electron transfer through complex IV is from cyt c to the CuA center, to the
heme a, to the a3- CuB and finally to the O2
• For every four electrons passing through this complex, the enzyme
consumes four “substrate” H+ from the matrix in converting O2 to 2H2O
• The enzyme also uses the energy of this redox reaction to pump protons
outward into the intermembrane space
• The process must occur without release of incompletely reduced
intermediates such as hydrogen peroxide or hydroxyl free radicals that
would damage cellular components

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Electron transfer begins when two
molecules of reduced cyt c each
donate an electron to a binuclear
center CuA. From here electrons pass
through heme a to the Fe-Cu center
(cyt a3 and CuB). Oxygen binds to
heme a3 and is reduced to its peroxy
derivative by the two electrons
from the Fe-Cu center. Delivery of two
more electrons from cyt c converts the
peroxyl derivative to two molecules of
water consuming four
“substrate”protons from the matrix. At
the same time four more protons are
pumped from the matrix.
Cyanide, azide and carbon
monoxide inhibit cytochrome
oxidase
Cyanide and azide react with the ferric form
of heme a3, whereas carbon monoxide
inhibit the ferrous form
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ATP synthesis
• Protons are translocated to the exterior of the inner mitochondrial membrane by
oxidation in the respiratory chain
– Since the membrane is impermeable to ions, protons accumulate outside the
membrane, creating an electrochemical potential difference across the
membrane
– electrochemical potential difference consists of
• Chemical potential difference (pH)
• Electrical potential difference (Charge)
• According to the chemiosmotic theory the electrochemical potential difference
generates a proton-motive force as protons flow back passively into the matrix
through a proton pore associated with ATP synthase. Its this proton-motive force that
drives the synthesis of ATP
A membrane–located ATP synthase functions as a Rotary Motor to form ATP
• The electrochemical potential difference is used to drive a membrane–located ATP
synthase which in the presence of Pi + ADP forms ATP
• Scattered over the surface of the inner membrane are the phosphorylating complexes
of ATP synthase, responsible for the production of ATP
• These consist of - F1 protein subunits which project into the matrix and
contain the phosphorylating mechanism
- F0, a disc of protein subunits that spans the membrane
-The F0 is connected to the F1 through the γ subunit
• Protons passing through the F0 dick causes it and the attached γ subunit to rotate
This rotation results in synthesis of ATP on the F1 sub complex. Three ATP
molecules are generated per revolution

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 F1

ATP
ADP + Pi

γ H+
Inside
Mitochondrial inner
membrane
C C C C
Outside F0

H+

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• Inhibitors of passage of electrons to O2 can block ATP synthesis. The converse is
true; inhibitors of ATP synthesis can block flow of electrons. Electron flow and ATP
synthesis are coupled; neither reaction occurs without the other
– This is explained by the chemiosmotic theory. When the flow of protons into the
matrix through the proton channel of ATP synthase is blocked (eg with
oligomycin), no path exists for the return of protons to the matrix and the
continued extrusion of protons by the activity of the respiratory chain generates a
large proton gradient. The proton gradient builds up until the free energy of
pumping protons against this gradient exceeds the energy released by the
transfer of electrons from NADH to O2.. At this point electron flow stops
• Certain conditions or chemical reagents can uncouple oxidation from phosphorylation
– Eg 2,4-dinitrophenol (DNP) has hydrophobic properties which allows it to
diffuse readily across mitochondrial membranes. Enters the mitochondrial matrix
in the protonated form. When the proton is released the proton gradient is
dissipated
– Ionophores such as valinomycin allow inorganic ions to pass easily through
membranes. Uncouple electron transfers from oxidative phosphorylation by
dissipating the electrical contribution to the electrochemical gradient
– Electron transport from NADH to O2 proceeds normally but ATP is not formed.
This leads to increased O2 consumption and oxidation of NADH
– Oxidation of brown fat in newborn mammals serves not to produce ATP but to
generate heat to keep the new born warm
– Mitochondria of brown fat have a unique protein in their inner membrane called
thermogenin (uncoupling protein)
• Thermogenin provides a path for the protons to return to the matrix without
passing through the FoF1 complex
• As a result the energy of oxidation is not conserved by ATP formation, but is
dissipated as heat
• The same principle applies in hibernating animals 21
• The protons pumped out per pair of electrons are 10 for NADH and 6 for
FADH2
• The number of protons required to drive the synthesis of an ATP molecule
is 4
• Therefore each NADH generates about 10/4 or 2.5 ATP molecules and
each FADH2 generates about 6/4 or 1.5 ATP molecules
• NB Values of 3.0 and 2.0 are used in some cases

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 The proton-motive force energizes active transport
• The proton-motive force also drives some processes essential for oxidative
phosphorylation
• Two systems transport ADP and Pi into the matrix and ATP out
Adenine
nucleotide
translocase Adenine nucleotide translocase
(adenine nucleotide transporter) transports
ATP4– ATP4–
ADP3– into the matrix in exchange for an
ADP3– ATP4– that is simultaneously transported
ADP3–
out. Because the antiporter moves 4
negative charges out for 3 negative
ATP 3H+ 3H+ charges that move in, its activity is
synthase favoured by the transmembrane
electrochemical gradient, which gives the
– – matrix a net negative charge; the proton-
H2PO4 H2PO4
motive force drives the ADP-ATP
exchange. Enzyme is inhibited by
H+ H+ atractyloside
Phosphate
translocase Phosphate translocase promotes
symport of one H2PO4– and one H+ into
the matrix. This too is favoured by the
transmembrane proton gradient
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Electrons from cytosolic NADH enter the mitochondria by shuttles
• The inner mitochondrial membrane is impermeable to NADH and NAD+
• NADH is formed by glycolysis in the cytosol and NAD+ must be regenerated for
glycolysis to continue.
• Electrons from NADH rather than NADH itself are carried across the mitochondrial
membrane.
 The glycerophosphate shuttle
– One carrier is glycerol 3-phosphate in the glycerophosphate shuttle which
readily traverses the outer mitochondrial membrane.
– Electrons are transferred from NADH to dihydroxyacetone phosphate to form
glycerol 3-phosphate. Catalyzed by cytosolic glycerol 3-phosphate
dehydrogenase.
– Glycerol 3-phosphate is reoxidized to dihydroxyacetone phosphate on outer
surface of inner mitochondrial membrane in a reaction catalyzed by
mitochondrial glycerol 3-phosphate dehydrogenase. In the process an electron
pair is transferred to FAD to form FADH2.
– Dihydroxyacetone phosphate formed in the oxidation of glycerol 3-phosphate
diffuses back into the cytosol.
– Since the glycerophosphate shuttle is linked to the flavoprotein rather than NAD+
only 1.5 rather than 2.5 molecules of ATP are formed.
– This shuttle is irreversible and operates in the muscle and brain

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 In the malate-aspartate shuttle
• It’s the most active NADH shuttle that functions in the liver, kidney and heart
mitochondria
• Electrons are transferred from cytosolic NADH in the cytosol to cytosolic
oxaloacetate, forming malate, which traverses the inner mitochondrial
membrane via the malate-α-ketoglutarate transporter and transfers the
electrons in the matrix to NAD+ forming NADH.
• The NADH can then pass on the electrons to the respiratory chain where
about 2.5 molecules of ATP are generated as the pair of electrons pass to
O2
• Resulting oxaloacetate does not readily cross the inner mitochondrial
membrane and a transamination reaction occurs to form aspartate which
can be transported via the glutamate-aspartate transporter to the cytosolic
side.
-In contrast to the glycerophosphate shuttle, this shuttle is reversible. NADH
can be brought into the mitochondria by the malate-aspartate shuttle only if
the NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial
matrix.

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Creatine phosphate shuttle facilitates transport of high-energy phosphate
from mitochondria
• ATP and ADP do not diffuse freely from the inner mitochondrial membrane
• A specific protein adenine nucleotide translocase or adenine nucleotide transporter
enables these highly charged molecules to traverse the permeability barrier
• Flows of ATP and ADP are coupled. ADP enters the mitochondrial matrix only when
ATP exits and vice versa.
• Atractyloside inhibits this transporter
• The shuttle acts as a dynamic system for transfer of high energy phosphate from the
mitochondria in active tissues like muscles.
• An isoenzyme of creatine kinase (CKm) found in the mitochondrial intermembrane
space , catalyzes the transfer of high energy phosphate from ATP emerging from the
adenine nucleotide transporter to creatineforming creatine phosphate
• In turn the creatine phosphate is transported into the cytosol via protein pores in the
outer membrane where it becomes available for generation of extramitochondrial
ATP.
• CKa, creatine kinase is concerned with large requirements for ATP eg muscle
contraction; CKc, creatine kinase for maintaining equilibrium between creatine and
creatine phosphate and ATP/ADP; CKg, creatine kinase for coupling glycolysis to
creatine phosphate synthesis

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 Energy- requiring
processes eg
ATP muscle contraction ADP

CKa

ATP ADP

Creatine Creatine phosphate

CKc

CKg ADP
ATP
Glycolysis

Outer mitochondrial
membrane
CKm

ATP ADP Inter-membrane

space

ANT

Oxidative
phosphorylation Inner mitochondrial
membrane
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Regulation of oxidative phosphorylation
• Oxidative phosphorylation produces most of the ATP made in aerobic cells
• Oxidative phosphorylation is regulated by cellular energy needs
• The rate of respiration in mitochondria is generally limited by the availability of Pi
acceptor, ADP - This is called the acceptor control of respiration
• Energy status of the cell can be measured by intracellular concentration of ADP or
the mass–action ratio of the ADP-ATP system i.e,[ATP]/([ADP][Pi])
• Normally this ratio is very high
• When the rate of some energy-requiring process (eg protein synthesis) increases, the
rate of breakdown of ATP to ADP and Pi increases lowering the mass action ratio
– With more ADP available for oxidative phosphorylation, the rate of respiration increased,
causing regeneration of ATP
– This continues until the mass-action ratio returns to its normal high level, at which point
respiration slows
– In short, ATP is formed only as fast as it is used in energy requiring processes

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• Briefly explain how the flow of electrons in the electron transport chain leads to
ATP synthesis
• What is an uncoupler? How do uncouplers effect electron transport chain and
oxidative phosphorylation?
• What is the rationale behind the use of uncoupler as slimming tablets?
• Where do electrons that enter the electron transport chain at the level of coenzyme
Q come from?
• Why is less ATP formed when electrons enter the electron transport chain at the
level of coenzyme Q than when entry is at the level of Complex I?
• What is meant by reduction potential?
• FMN and Coenzyme Q differ from NADH and cytochromes as electron
acceptors/carriers. Briefly explain this difference. (no structures are necessary).
• Briefly describe the chemiosmotic hypothesis for oxidative phosphorylation.

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